Methods for generating recombinant adeno-associated virus viral particles

Abstract

Provided herein are improved methods for producing recombinant viral particles. In some embodiments, the methods for producing recombinant viral particles described herein include providing a suspension cell culture comprising a population of cells capable of producing viral particles, transferring a first volume of polynucleotide:transfection reagent complexes to the suspension culture for transfecting the cells, and transferring a second volume of polynucleotide:transfection reagent complexes to the suspension culture, wherein the transferring of the first and second volumes of polynucleotide:transfection reagent complexes is performed simultaneously or sequentially in any order. In some embodiments, the recombinant viral particles are recombinant AAV (rAAV) particles.

Description

Technical Field

[0001] The present disclosure relates to a method for generating recombinant virus particles in large-scale suspension cell cultures, which includes transfecting cells in the culture.

[0002] Cross-reference to Related Applications This application claims the benefit of U.S. Patent Application No. 63 / 126,405, filed on December 16, 2020, which is hereby incorporated by reference in its entirety.

Background Art

[0003] Background Recombinant adeno-associated virus (AAV)-based vectors are currently the most widely used developing gene therapy products. The reasons for the preference for the use of rAAV vector systems are, in part, the absence of diseases associated with wild-type viruses, the ability of AAV transduction in both non-dividing and dividing cells, and the observation of long-term robust transgene expression in clinical trials, which indicates great potential for delivery in gene therapy applications. In addition, various natural and recombinant rAAV vector serotypes specifically target various tissues, organs, and cells and help avoid any existing immunity to the vector, thus expanding the therapeutic applications of AAV-based gene therapy. Before recombinant virus particles can be more widely adopted in late clinical stages and commercial use, there is a need to develop new methods for the large-scale production of recombinant virus particles.

[0004] Therefore, in the art, there is a need to improve the productivity and yield of methods for generating rAAV particles on a large scale.

Summary of the Invention

[0005] Brief Summary In one embodiment, the disclosure provides a method for isolating recombinant adeno-associated virus (rAAV) genomes using size exclusion chromatography. In some embodiments, the method comprises subjecting a composition containing rAAV particles to conditions that denature the rAAV particles, and then subjecting the composition containing the denatured rAAV particles to size exclusion chromatography. In some embodiments, the mobile phase of the size exclusion chromatography comprises a salt, an organic solvent, or a washing agent. In some embodiments, the mobile phase further comprises a buffer. In some embodiments, rAAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV. It contains capsid proteins of PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 serotypes. In some embodiments, rAAV contains capsid proteins of AAV8 or AAV9 serotypes.

[0006] In further embodiments, the disclosure provides a method for characterizing recombinant adeno-associated virus (rAAV) particles using size exclusion chromatography. Characterization of isolated rAAV particles includes, but is not limited to, quantifying the vector genome size purity of a composition containing isolated rAAV particles, evaluating the folding or secondary structure of the vector genome within the capsid, and quantifying the vector genome titer (Vg) of a composition containing isolated rAAV particles. In some embodiments, the mobile phase of the size exclusion chromatography comprises a salt, an organic solvent, or a washing agent. In some embodiments, the mobile phase further comprises a buffer. In some embodiments, rAAV particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV It contains capsid proteins of serotypes PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16. In some embodiments, rAAV contains capsid proteins of serotypes AAV8 or AAV9.

[0007] In some embodiments, the methods disclosed herein are suitable for batch release, such as batch release testing and / or lot release testing. In some embodiments, the methods disclosed herein are carried out as part of lot release testing.

[0008] In some embodiments, this disclosure provides the following:

[0009] [1.] A method for generating recombinant virus particles, a) Prepare a suspension cell culture in a volume of approximately 200 liters to approximately 20,000 liters containing a population of cells capable of producing the recombinant virus particles, b) Mixing one or more polynucleotides with at least one translocation reagent to form a first mixture, incubating the mixture to form a polynucleotide:translocation reagent complex, and transferring the polynucleotide:translocation reagent complex to the suspension culture for translocation into the cells. c) Mixing one or more polynucleotides with at least one translocation reagent to form a second mixture, incubating the mixture to form a polynucleotide:translocation reagent complex, and transferring the polynucleotide:translocation reagent complex to the suspension culture for translocation into the cells. d) Maintaining the cell culture containing the transfused cells under conditions that enable the generation of the recombinant virus particles. Includes, (i) The one or more polynucleotides include genes necessary for the generation of recombinant virus particles, (ii) The mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 60 minutes. (iii) The transfer in step b) and step c) is carried out over a period of time of approximately 6 hours or less, and (iv) The transfers in steps b) and c) are performed simultaneously or sequentially in any order. The aforementioned method.

[0010] [2.] The method described in [1], wherein step c) is repeated once more.

[0011] [3.] The method according to [1], wherein step c) is repeated one or more times.

[0012] [4.] The method according to [1], wherein step c) is repeated 1, 2, 3, 4, 5, 6, 7, or 8 times.

[0013] [5.] The method according to any one of [1] to [4], wherein the combined volume of the polynucleotide:transfer reagent complex transferred to the suspension culture is between approximately 5% and approximately 20% of the volume of the suspension cell culture in step a).

[0014] [6.] The method according to any one of [1] to [5], wherein the transfer in step c) is started between approximately 5 minutes and approximately 60 minutes after the transfer in step b) is completed.

[0015] [7.] The method according to any one of [1] to [5], wherein the transfer in step c) is started within approximately 60 minutes after the transfer in step b) is completed.

[0016] [8.] A method for increasing the generation of recombinant virus particles, a) Prepare a suspension cell culture in a volume of approximately 200 liters to approximately 20,000 liters containing a population of cells capable of producing the recombinant virus particles, b) Mixing one or more polynucleotides with at least one translocation reagent to form a first mixture, incubating the mixture to form a polynucleotide:translocation reagent complex, and transferring the polynucleotide:translocation reagent complex to the suspension culture for translocation into the cells. c) Mixing one or more polynucleotides with at least one translocation reagent to form a second mixture, incubating the mixture to form a polynucleotide:translocation reagent complex, and transferring the polynucleotide:translocation reagent complex to the suspension culture for translocation into the cells. d) Maintaining the cell culture containing the transfused cells under conditions that enable the generation of the recombinant virus particles. Includes, (i) The one or more polynucleotides contain genes necessary for the production of recombinant virus particles, (ii) The mixing, incubating, and transferring in steps b) and c) are each completed in less than about 60 minutes, (iii) The transferring in steps b) and c) is carried out over a period of about 6 hours or less, and (iv) The transferring in steps b) and c) is carried out continuously either simultaneously or in any order, The method.

[0017] [9.] The method according to [8], wherein step c) is repeated once more.

[0018] [10.] The method according to [8], wherein step c) is repeated one or more times.

[0019] [11.] The method according to [8], wherein step c) is repeated 1, 2, 3, 4, 5, 6, 7, or 8 times.

[0020] [12.] The combined volume of the polynucleotide: transfection reagent complex transferred to the suspension culture is between about 5% and about 20% of the volume of the suspension cell culture in step a), according to any one of [8] to

[11] .

[0021] [13.] The transferring in step c) is started between about 5 minutes and about 60 minutes after completion of the transferring in step b), according to any one of [8] to

[12] .

[0022] [14.] The transferring in step c) is started within about 60 minutes after completion of the transferring in step b), according to any one of [8] to

[13] .

[0023] [15.] The mixing of the one or more polynucleotides with at least one transfection reagent is carried out by an in-line mixer, according to any one of [1] to

[14] .

[0024] [16.] The method according to any one of [1] to

[15] , wherein the mixing, incubation, and transfer in steps b) and c) are each completed in less than 30 minutes.

[0025] [17.] The method according to any one of [1] to

[15] , wherein the mixing, incubation, and transfer of steps b) and c) are each completed in less than approximately 35 minutes.

[0026] [18.] The method according to any one of [1] to

[17] , wherein the incubation in step b) and step c) is each about 10 to about 20 minutes.

[0027] [19.] The method according to any one of [1] to

[17] , wherein the incubation in step b) and step c) is each about 10 to about 15 minutes.

[0028] [20.] The method according to any one of [1] to

[19] , wherein the at least one trait transfer reagent comprises a stable cationic polymer.

[0029] [21.] The method according to any one of [1] to

[20] , wherein the at least one translocation reagent comprises PEI.

[0030] [22.] The method according to any one of [1] to

[21] , wherein the cell culture has a volume between approximately 200 liters and approximately 5,000 liters.

[0031] [23.] The method according to any one of [1] to

[21] , wherein the cell culture has a volume between approximately 200 liters and approximately 2,000 liters.

[0032] [24.] The method according to any one of [1] to

[21] , wherein the cell culture has a volume between approximately 200 liters and approximately 1,000 liters.

[0033] [25.] The method according to any one of [1] to

[21] , wherein the cell culture has a volume between approximately 200 liters and approximately 500 liters.

[0034] [26.] The method according to any one of [1] to

[21] , wherein the cell culture has a volume of approximately 200 liters, approximately 300 liters, approximately 400 liters, approximately 500 liters, approximately 750 liters, approximately 1,000 liters, approximately 2,000 liters, approximately 3,000 liters, or approximately 5,000 liters.

[0035] [27.] The method according to any one of [1] to

[21] , wherein the cell culture has a volume of approximately 500 liters.

[0036] [28.] The method according to any one of [1] to

[21] , wherein the cell culture has a volume of approximately 1,000 liters.

[0037] [29.] The method according to any one of [1] to

[21] , wherein the cell culture has a volume of approximately 2,000 liters.

[0038] [30.] The method according to any one of [1] to

[29] , wherein the population of cells comprises a population of mammalian cells or insect cells.

[0039] [31.] The method according to any one of [1] to

[29] , wherein the population of cells comprises a population of mammalian cells.

[0040] [21.] The method according to any one of [1] to

[29] , wherein the population of cells comprises a population of HEK293 cells, HEK-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, and / or PerC6 cells.

[0041] [33.] The method according to any one of [1] to

[29] , wherein the population of cells comprises a population of HEK293 cells.

[0042] [34.] The method according to any one of [1] to

[33] , wherein the suspension cell culture prepared in step a) contains between approximately 2 × 10⁶ and approximately 10⁷ viable cells / ml.

[0043] [35.] The method according to any one of [1] to

[34] , wherein the cell culture is maintained under conditions that enable the generation of recombinant virus particles for a period of about 2 to about 10 days, about 3 to about 5 days, or about 5 to 14 days.

[0044] [36.] The method according to any one of [1] to

[34] , wherein the cell culture is maintained for approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, or approximately 7 days.

[0045] [37.] The method according to any one of [1] to

[34] , wherein the cell culture is maintained for at least about 3 days.

[0046] [38.] The method according to any one of [1] to

[34] , wherein the cell culture is maintained for approximately 3 days.

[0047] [39.] The method according to any one of [1] to

[34] , wherein the cell culture is maintained for approximately 4 days.

[0048] [40.] The method according to any one of [1] to

[39] , wherein the recombinant virus particle is a recombinant adeno-associated virus (rAAV) particle or a recombinant lentivirus particle.

[0049] [41.] The method according to any one of [1] to

[39] , wherein the recombinant virus particle is an rAAV particle.

[0050] [42.] The rAAV particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AA V.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AA The method according to

[41] , comprising a capsid protein of serotype V2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[0051] [43.] The method according to

[41] , wherein the rAAV particles comprise a capsid protein of the AAV8, AAV9, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, or AAV.hu37 serotype.

[0052] [44.] The method according to

[41] , wherein the rAAV particles comprise a capsid protein of the AAV8 or AAV9 serotype.

[0053] [45.] The method according to any one of

[41] to

[44] , wherein the rAAV particle comprises a genome containing a transgene.

[0054] [46.] The method according to

[45] , wherein the introduced gene comprises a regulatory element responsively linked to a polynucleotide encoding a polypeptide.

[0055] [47.] The method according to

[46] , wherein the regulatory element comprises one or more of an enhancer, a promoter, and a poly-A region.

[0056] [48.] The method according to

[45] or

[46] , wherein the regulatory element and the polynucleotide encoding the polypeptide are heterogeneous.

[0057] [49.] The method according to any one of

[45] to

[48] , wherein the introduced gene encodes an antibody or an antigen-binding fragment thereof, a fusion protein, an Fc fusion polypeptide, an immunoadhesin, an immunoglobulin, a modified protein, a protein fragment, or an enzyme.

[0058] [50.] The method according to any one of

[45] -

[48] , wherein the transgene encodes anti-VEGF Fab, idulonidase (IDUA), iduronate 2-sulfatase (IDS), low-density lipoprotein receptor (LDLR), tripeptidyl peptidase 1 (TPP1), or a non-membrane-bound splice variant of VEGF receptor 1 (sFlt-1).

[0059] [51.] The transgenes include gamma-sarcoglycan, Rab escort protein 1 (REP1 / CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide gate channel alpha-3 (CNGA3), cyclic nucleotide gate channel beta-3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosome-associated membrane protein 2 isoform B (LAMP2B), factor VIII, factor IX, retinitis pigmentosa GTPase regulator (RPGR), retinoskin (RS1), and sarcoplasmic reticulum kahl. Cium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamate decarboxylase (GAD), glial cell line-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, microdystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) , arylsulfatase B (ARSB), N-acetyl-alpha-glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha-1-antitrypsin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN1), survival motor neuron (SMN2), neurturin (NRTN), neurotroph The method according to any one of

[45] -

[48] , encoding fin-3 (NT-3 / NTF3), porphobilinogen deaminase (PBGD), nerve growth factor (NGF), mitochondrial code NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dysferrin, MER proto-oncogene tyrosine kinase (MERTK), cystic fibrosis membrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgG1) Fc fusion.

[0060] [52.] The one or more polynucleotides described above a) rAAV genome to be packaged, b) Adenovirus helper function required for packaging, c) Sufficient AAV rep protein for packaging, and d) Sufficient AAV cap protein for packaging A method of coding one of

[41] to

[51] .

[0061] [53.] The method according to

[52] , wherein the one or more polynucleotides comprises a polynucleotide encoding the rAAV genome, a polynucleotide encoding the AAV rep protein and the AAV cap protein, and a polynucleotide encoding the adenovirus helper function.

[0062] [54.] The method according to either

[52] or

[53] , wherein the adenovirus helper function comprises at least one of the adenovirus E1a gene, E1b gene, E4 gene, E2a gene, and VA gene.

[0063] [55.] The method according to any one of

[0412] to

[54] , further comprising recovering the rAAV particles.

[0064] [56.] The method according to any one of

[41] to

[55] , wherein the cell culture generates rAAV particles between approximately 5 × 10 e + 10 GC / ml and approximately 1 × 10 e + 12 GC / ml.

[0065] [57.] The method according to any one of

[41] to

[56] , wherein the cell culture produces at least about twice as many rAAV particles as measured as GC / ml as the reference method, which comprises a single step of mixing, incubating and transferring the same volume of polynucleotide:transfer reagent complex.

[0066] Isolated rAAV particles produced by any one of the methods described in

[58] ,

[41] , and

[57] . A composition containing the following:

[0067] Further other features and advantages of the compositions and methods described herein will become clearer from the following detailed description when read in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0068] [Figure 1] Flowchart of the complex preparation process for a single-dose 50L transient transgenic transfer. [Figure 2] Flowchart of the complex preparation process for a single dose of 200L transient transgenic transfer. [Figure 3] Flowchart of the complex preparation process for a single dose of 500L transient transgenic transfer. [Figure 4] Bioreactor productivity of 50L, 200L, and 500L reactors using single-dose transfusion. [Figure 5] As the complex formation time increases, the translocation complex becomes larger. [Figure 6] A flowchart of the complex preparation process for testing the effect of complex formation time on transient translocation efficiency. [Figure 7] The effect of complex formation time on transient translocation efficiency. [Figure 8] Flowchart of the complex preparation process for 200L fractional transient transfusion. [Figure 9] Productivity comparison between single-dose transfusion and segmented transfusion in a 200L bioreactor. [Figure 10] Robustness of fragmentation of traits. [Figure 11] Flowchart of the complex preparation process for 500L fractional transient transfusion. [Figure 12] Bioreactor productivity of a 500L reactor using segmented transfusion. [Figure 13]Flowchart of the segmented trait transfer process in a 2,000L process scale-down to 200L. [Modes for carrying out the invention]

[0069] Detailed explanation This specification provides a method for generating recombinant virus particles in a large-scale culture, such as a suspension culture, comprising transferring more than one volume of separately generated compositions containing a polynucleotide:transfer reagent complex to the culture for translocation into cells, wherein the transfer of more than one volume of compositions is carried out over a period of about six hours or less, and the transfer of more than one volume of compositions is carried out simultaneously or sequentially. In some embodiments, the transfer reagent comprises a stable cationic polymer (e.g., PEI). In some embodiments, the large-scale cell culture is between about 200 liters and about 20,000 liters, and the combined volume of separately generated compositions containing the polynucleotide:transfer reagent complex transferred to the culture is between about 5% and about 20% of the volume of the cell culture. In some embodiments, each volume of separately generated compositions containing the polynucleotide:transfer reagent complex is generated and transferred to the cell culture in about 60 minutes or less (e.g., about 30 minutes or less). In some embodiments, the combined volume of separately produced compositions containing the polynucleotide:transfer reagent complex is larger than the volume that can be produced in a single batch in about 60 minutes or less (e.g., about 30 minutes or less) and transferred to a large culture. While not bound by any particular theory, the methods disclosed herein result in increased productivity by enabling the transfer of a large total volume of composition containing the polynucleotide:transfer reagent complex to a cell culture, where each component volume of the composition is produced in about 60 minutes or less (e.g., about 30 minutes or less) and transferred to the cell culture. In some embodiments, each volume of separately produced compositions containing the polynucleotide:transfer reagent complex is produced in 60 minutes or less, 50 minutes or less, 40 minutes or less, 35 minutes or less, 30 minutes or less, 25 minutes or less, or 20 minutes or less and transferred to the cell culture. In some embodiments, each volume of separately produced compositions containing the polynucleotide:transfer reagent complex is produced in 30 minutes or less and transferred to the cell culture. In some embodiments, the productivity of the methods disclosed herein is at least about twice that of a reference method, which involves transferring the same total volume of a composition containing a polynucleotide:transfer reagent complex produced in a single batch.In some embodiments, productivity is quantified as virus particles per ml of culture at collection. In some embodiments, productivity is quantified as the number of virus particles recovered from a unit volume, e.g., 1 ml of culture. In some embodiments, the cell culture is a suspension cell culture. In some embodiments, the cell culture contains adherent cells that grow attached to microcarriers or macrocarriers in a stirred bioreactor. In some embodiments, the cell culture is a suspension cell culture containing HEK293 cells.

[0070] In some embodiments, the recombinant virus particles are recombinant adeno-associated virus (rAAV) particles. In some embodiments, rAAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV. It contains capsid proteins of PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 serotypes. In some embodiments, rAAV contains capsid proteins of AAV8 or AAV9 serotypes.

[0071] Given that a very large number of rAAV particles are required to prepare a single therapeutic dose, a doubling of rAAV yield would significantly reduce the product cost per unit dose. Increased virus yield would also reduce the cost of consumables needed to produce rAAV particles, as well as the capital investment costs associated with constructing industrial virus purification facilities.

[0072] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which this disclosure relates. To facilitate understanding of the methods of this disclosure, several terms and expressions are defined below.

[0073] For example, modifying the amount of a component in a composition, the concentration of a component in a composition, the flow rate, the rAAV particle yield, the feed volume, the salt concentration, similar values, and ranges thereof, “about” as used in the methods provided herein means variations in numerical quantities that may result from, for example, typical measurement and handling procedures used to prepare a concentrate or working solution; accidental errors in these procedures; differences in the manufacture, source, or purity of the components used in preparing the composition or performing the method; and similar considerations. The term “about” also includes amounts that differ as a composition or mixture having a particular initial concentration ages. The term “about” also includes amounts that differ as a composition or mixture having a particular initial concentration is mixed or processed. Whether modified by the term “about,” a claim includes an equivalent of that quantity. In some embodiments, the term “about” means a range of about 10 to 20 percent more or less than the number or range indicated. In further embodiments, “about” means plus or minus 10 percent of the number or range indicated. For example, “about 10%” indicates a range of 9% to 11%.

[0074] "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its modifiers, derivatives, or pseudotypes. Unless otherwise required, the term encompasses all subtypes, as well as both the native and recombinant forms. The abbreviation "rAAV" refers to recombinant adeno-associated virus. The term "AAV" includes AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV, as well as their modifiers, derivatives, or pseudotypes. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, and "bovine AAV" refers to AAV that infects bovine mammals, and so on.

[0075] The term "recombination" as applied to AAV particles means that the AAV particle is the product of one or more procedures that result in an AAV particle construct that is essentially different from the AAV particle itself.

[0076] Recombinant adeno-associated virus particles, or "rAAV particles," refer to viral particles composed of at least one AAV capsid protein and a capsidized polynucleotide rAAV vector genome containing heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, e.g., a transgene to be delivered to mammalian cells). rAAV particles can be any AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10, or their derivatives / modifiers / pseudotypes), including any modifiers, derivatives, or pseudotypes. Such AAV serotypes and derivatives / modifiers / pseudotypes, as well as methods for generating such serotypes / derivatives / modifiers / pseudotypes, are known in the art (see, for example, Asokan et al., Mol.Ther. 20(4):699-708 (2012)).

[0077] The rAAV particles of this disclosure may be any serotype or any combination of serotypes (for example, a population of rAAV particles containing two or more serotypes (for example, two or more of rAAV2, rAAV8, and rAAV9 particles)). In some embodiments, the rAAV particles are rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, or other rAAV particles, or two or more combinations thereof. In some embodiments, the rAAV particles are rAAV8 or rAAV9 particles.

[0078] In some embodiments, the rAAV particles have an AAV capsid protein of a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, or derivatives, modifiers, or pseudotypes thereof. In some embodiments, the rAAV particles have an AAV capsid protein of a serotype consisting of AAV8, AAV9, or derivatives, modifiers, or pseudotypes thereof.

[0079] The term “cell culture” means cells grown in suspension or attached to microcarriers or macrocarriers, bioreactors, roller bottles, hyperstacks, microspheres, macrospheres, flasks, etc., and the components of the supernatant or suspension itself, which include, but are not limited to, rAAV particles, cells, cell debris, cell contaminants, colloidal particles, biomolecules, host cell proteins, nucleic acids, lipids, and flocculants. Large-scale approaches such as bioreactors (including suspension cultures and adherent cells grown attached to microcarriers or macrocarriers in agitated bioreactors) are encompassed by the term “cell culture.” Cell culture procedures for both large-scale and small-scale production of viral particles or proteins are also encompassed by this disclosure. In some embodiments, the term “cell culture” means cells grown in suspension. In some embodiments, the term “cell culture” means adherent cells grown attached to microcarriers or macrocarriers in agitated bioreactors.

[0080] As used herein, the terms “purify,” “separate,” “isolate,” “remove,” “isolate,” or “isolate” mean increasing the degree of purity of a target product (e.g., rAAV particles and rAAV genome) from a sample containing the target product and one or more impurities. Typically, the degree of purity of a target product is increased by removing (completely or incompletely) at least one impurity from the sample. In some embodiments, the degree of purity of rAAV in a sample is increased by removing (completely or incompletely) one or more impurities from the sample using the methods described herein.

[0081] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural form unless otherwise explicitly stated in the context.

[0082] Whenever embodiments described herein using the phrase "including" should be understood as also providing other similar embodiments described in terms of "consisting of" and / or "essentially consisting of".

[0083] When the term "and / or" is used herein in expressions such as "A and / or B," it is intended to include both A and B, A or B, A (alone), and B (alone). Similarly, when the term "and / or" is used in expressions such as "A, B, and / or C," it is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0084] Where embodiments of this disclosure describe the Markush Group or other alternative groups, the methods of this disclosure encompass not only the entire group as a whole, but also each individual member of the group, all possible subgroups of the main group, and even the main group in which one or more group members are absent. The methods of this disclosure also anticipate the express exclusion of one or more group members in the methods of this disclosure.

[0085] Method for generating recombinant viruses In some embodiments, the present disclosure relates to a method for generating recombinant virus particles, a) Prepare a suspension cell culture of approximately 200 liters to approximately 20,000 liters containing a population of cells capable of producing recombinant virus particles, b) To form a first mixture, one or more polynucleotides are mixed with at least one translocation reagent; the mixture is incubated to form a polynucleotide:translocation reagent complex; and the polynucleotide:translocation reagent complex is transferred to a suspension culture for translocation into cells. c) To form a second mixture, one or more polynucleotides are mixed with at least one translocation reagent; the mixture is incubated to form a polynucleotide:translocation reagent complex; and the polynucleotide:translocation reagent complex is transferred to a suspension culture for translocation into cells. d) Maintain the cell culture containing transfected cells under conditions that enable the generation of recombinant virus particles. Includes, The present invention provides a method in which one or more polynucleotides contain genes necessary for the generation of recombinant viral particles. In some embodiments, the one or more polynucleotides contain one or more helper genes, rep genes, cap genes, and transgenes (e.g., the target gene or the rAAV genome to be packaged). In some embodiments, the combined volume of the polynucleotide:transfer reagent complex transferred to the suspension culture is between about 5% and about 20% of the volume of the cell culture in step a). In some embodiments, the combined volume of the polynucleotide:transfer reagent complex transferred to the suspension culture is between about 7% and about 15% of the volume of the cell culture in step a). In some embodiments, the combined volume of the polynucleotide:transfer reagent complex transferred to the suspension culture is about 10% of the volume of the cell culture in step a). In some embodiments, the transfer in step c) is started before the transfer in step b) is completed. In some embodiments, the transfer in step c) is started immediately after the transfer in step b) is completed. In some embodiments, the transfer in step c) is initiated between approximately 5 and 60 minutes after the completion of the transfer in step b). In some embodiments, the transfer in step c) is initiated within approximately 5 minutes after the completion of the transfer in step b). In some embodiments, the transfer in step c) is initiated within approximately 10 minutes after the completion of the transfer in step b). In some embodiments, the transfer in step c) is initiated within approximately 15 minutes after the completion of the transfer in step b). In some embodiments, the transfer in step c) is initiated within approximately 20 minutes after the completion of the transfer in step b). In some embodiments, the transfer in step c) is initiated within approximately 30 minutes after the completion of the transfer in step b). In some embodiments, the transfer in step c) is initiated within approximately 45 minutes after the completion of the transfer in step b). In some embodiments, the transfer in step c) is initiated within approximately 60 minutes after the completion of the transfer in step b).In some embodiments, the mixing, incubation, and transfer in steps b) and c) are completed in less than approximately 90 minutes, less than approximately 60 minutes, less than approximately 50 minutes, less than approximately 40 minutes, less than approximately 35 minutes, less than approximately 30 minutes, less than approximately 25 minutes, or less than approximately 20 minutes, respectively. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are completed in less than approximately 60 minutes, respectively. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are completed in less than approximately 50 minutes, respectively. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are completed in less than approximately 40 minutes, respectively. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are completed in less than approximately 35 minutes, respectively. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 30 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 25 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 20 minutes. In some embodiments, the incubation in steps b) and c) is performed between approximately 5 minutes and 20 minutes, between approximately 10 minutes and 20 minutes, between approximately 5 minutes and 15 minutes, between approximately 10 minutes and 15 minutes, or between approximately 15 minutes and 20 minutes. In some embodiments, the incubation in steps b) and c) is performed between approximately 10 minutes and 15 minutes. In some embodiments, the incubation in steps b) and c) is performed for about 5 minutes, about 10 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, or about 20 minutes. In some embodiments, the incubation in steps b) and c) is performed for about 10 minutes.In some embodiments, incubation in steps b) and c) is performed for about 12 minutes. In some embodiments, incubation in steps b) and c) is performed for about 15 minutes. In some embodiments, the transfer in steps b) and c) is performed over a period of about 1 to 12 hours. In some embodiments, the transfer in steps b) and c) is performed over a period of about 1 to 8 hours. In some embodiments, the transfer in steps b) and c) is performed over a period of about 1 to 6 hours. In some embodiments, the transfer in steps b) and c) is performed over a period of about 1 to 5 hours. In some embodiments, the transfer in steps b) and c) is performed over a period of about 1 to 4 hours. In some embodiments, the transfer in steps b) and c) is performed over a period of about 1 to 3 hours. In some embodiments, the transfer in steps b) and c) is performed over a period of about 12 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 9 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 8 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 7 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 6 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 5 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 5 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 3 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 2 hours or less.In some embodiments, the transfer in steps b) and c) is carried out simultaneously or sequentially in any order. In some embodiments, the transfer in steps b) and c) is carried out sequentially in any order. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are carried out in the same manner. In some embodiments, the cell culture is a suspension culture. In some embodiments, the cell culture contains HEK293 cells adapted for growth in suspension culture. In some embodiments, the cell culture has a volume between about 400 liters and about 5,000 liters. In some embodiments, the cell culture has a volume of about 500 liters. In some embodiments, the cell culture has a volume of about 2,000 liters. In some embodiments, the translocation reagent contains a cationic polymer. In some embodiments, the translocation reagent contains PEI. In some embodiments, the recombinant virus particles are recombinant adeno-associated virus (rAAV) particles. In some embodiments, one or more polynucleotides comprise a mixture of three polynucleotides (one encoding cap and rep genes, one encoding adenovirus helper functions necessary for packaging (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and one encoding the rAAV genome to be packaged). In some embodiments, the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV particles have an AAV capsid protein of a serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles have AAV capsid proteins with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0086] In some embodiments, the present disclosure relates to a method for generating recombinant virus particles, a) Prepare a suspension cell culture of approximately 200 liters to approximately 20,000 liters containing a population of cells capable of producing recombinant virus particles, b) To form a first mixture, one or more polynucleotides are mixed with at least one translocation reagent; the mixture is incubated to form a polynucleotide:translocation reagent complex; and the polynucleotide:translocation reagent complex is transferred to a suspension culture for translocation into cells. c) To form a second mixture, one or more polynucleotides are mixed with at least one translocation reagent; the mixture is incubated to form a polynucleotide:translocation reagent complex; and the polynucleotide:translocation reagent complex is transferred to a suspension culture for translocation into cells. d) Maintain the cell culture containing transfected cells under conditions that enable the generation of recombinant virus particles. Includes, A method is provided in which one or more polynucleotides contain genes necessary for the generation of recombinant viral particles, and step c) is repeated one or more times. In some embodiments, step c) is repeated one more time. In some embodiments, step c) is repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, step c) is repeated twice. In some embodiments, step c) is repeated three times. In some embodiments, step c) is repeated four times. In some embodiments, step c) is repeated five times. In some embodiments, step c) is repeated six times. In some embodiments, step c) is repeated seven times. In some embodiments, step c) is repeated eight times. In some embodiments, step c) is repeated seven times. In some embodiments, step c) is repeated nine times. In some embodiments, step c) is repeated seven times. In some embodiments, step c) is repeated ten times. In some embodiments, the combined volume of the polynucleotide:transfer reagent complex transferred to the suspension culture is between approximately 5% and approximately 20% of the volume of the cell culture in step a). In some embodiments, the combined volume of the polynucleotide:transfer reagent complex transferred to the suspension culture is between approximately 7% and approximately 15% of the volume of the cell culture in step a). In some embodiments, the combined volume of the polynucleotide:transfer reagent complex transferred to the suspension culture is between approximately 10% of the volume of the cell culture in step a). In some embodiments, the transfer in step c) is started before the transfer in the preceding step is completed. In some embodiments, the transfer in step c) is started immediately after the transfer in the preceding step is completed. In some embodiments, the transfer in step c) is started between approximately 5 minutes and approximately 60 minutes after the transfer in the preceding step is completed. In some embodiments, the transfer in step c) is started within approximately 5 minutes after the transfer in the preceding step is completed. In some embodiments, the transfer in step c) is initiated within approximately 10 minutes after the completion of the transfer in the preceding step.In some embodiments, the transfer in step c) is initiated within approximately 15 minutes after the completion of the transfer in the preceding step. In some embodiments, the transfer in step c) is initiated within approximately 20 minutes after the completion of the transfer in the preceding step. In some embodiments, the transfer in step c) is initiated within approximately 30 minutes after the completion of the transfer in the preceding step. In some embodiments, the transfer in step c) is initiated within approximately 45 minutes after the completion of the transfer in the preceding step. In some embodiments, the transfer in step c) is initiated within approximately 60 minutes after the completion of the transfer in the preceding step. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are completed in less than approximately 90 minutes, less than approximately 60 minutes, less than approximately 50 minutes, less than approximately 40 minutes, less than approximately 35 minutes, less than approximately 30 minutes, less than approximately 25 minutes, or less than approximately 20 minutes, respectively. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are completed in less than approximately 60 minutes, respectively. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 50 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 40 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 35 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 30 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 25 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 20 minutes.In some embodiments, incubation in steps b) and c) is performed for about 5 to about 20 minutes, about 10 to about 20 minutes, about 5 to about 15 minutes, about 10 to about 15 minutes, or about 15 to about 20 minutes. In some embodiments, incubation in steps b) and c) is performed for about 10 to about 15 minutes. In some embodiments, incubation in steps b) and c) is performed for about 5 minutes, about 10 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, or about 20 minutes. In some embodiments, incubation in steps b) and c) is performed for about 10 minutes. In some embodiments, incubation in steps b) and c) is performed for about 12 minutes. In some embodiments, incubation in steps b) and c) is performed for about 15 minutes. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 1 to 12 hours. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 1 to 8 hours. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 1 to 6 hours. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 1 to 5 hours. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 1 to 4 hours. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 1 to 3 hours. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 12 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 9 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 8 hours or less. In some embodiments, the transfer of steps b) and c) is carried out over a period of time of approximately 7 hours or less.In some embodiments, the transfer in steps b) and c) is carried out over a period of about 6 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 5 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 5 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 3 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 2 hours or less. In some embodiments, the transfer in steps b) and c) is carried out simultaneously or sequentially in any order. In some embodiments, the transfer in steps b) and c) is carried out sequentially in any order. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are carried out in the same manner. In some embodiments, the cell culture is a suspension culture. In some embodiments, the cell culture contains HEK293 cells adapted for growth in suspension culture. In some embodiments, the cell culture has a volume between about 400 liters and about 5,000 liters. In some embodiments, the cell culture has a volume of about 500 liters. In some embodiments, the cell culture has a volume of about 2,000 liters. In some embodiments, the translocation reagent comprises a cationic polymer. In some embodiments, one or more polynucleotides comprise one or more helper genes, rep genes, cap genes, and transgenes (e.g., target genes). In some embodiments, the translocation reagent comprises PEI. In some embodiments, the recombinant virus particles are recombinant adeno-associated virus (rAAV) particles.In some embodiments, one or more polynucleotides comprise a mixture of three polynucleotides (one encoding cap and rep genes, one encoding adenovirus helper functions necessary for packaging (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and one encoding the rAAV genome to be packaged). In some embodiments, the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV particles have an AAV capsid protein of a serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles have AAV capsid proteins with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0087] In some embodiments, the present disclosure provides a method for increasing the generation of recombinant virus particles, a) Prepare a suspension cell culture of approximately 200 liters to approximately 20,000 liters containing a population of cells capable of producing recombinant virus particles, b) To form a first mixture, one or more polynucleotides are mixed with at least one translocation reagent; the mixture is incubated to form a polynucleotide:translocation reagent complex; and the polynucleotide:translocation reagent complex is transferred to a suspension culture for translocation into cells. c) To form a second mixture, one or more polynucleotides are mixed with at least one translocation reagent; the mixture is incubated to form a polynucleotide:translocation reagent complex; and the polynucleotide:translocation reagent complex is transferred to a suspension culture for translocation into cells. d) Maintain the cell culture containing transfected cells under conditions that enable the generation of recombinant virus particles. Includes, A method is provided in which one or more polynucleotides contain genes necessary for the generation of recombinant viral particles. In some embodiments, the method produces at least about twice as many rAAV particles, measured as GC / ml, compared to a reference method comprising a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfer reagent complex. In some embodiments, the method increases rAAV production by at least about 50%, at least about 75%, or at least about 100% compared to a reference method comprising a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfer reagent complex. In some embodiments, the method disclosed herein increases rAAV production by at least about 2 times, at least about 3 times, or at least about 5 times compared to a reference method comprising a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfer reagent complex. In some embodiments, the combined volume of polynucleotide:transfer reagent complex transferred to the suspension culture is between about 5% and about 20% of the volume of the cell culture in step a). In some embodiments, the combined volume of the polynucleotide:transfer reagent complex transferred to the suspension culture is between approximately 7% and 15% of the volume of the cell culture in step a). In some embodiments, the combined volume of the polynucleotide:transfer reagent complex transferred to the suspension culture is between approximately 10% of the volume of the cell culture in step a). In some embodiments, the transfer in step c) is started before the transfer in step b) is completed. In some embodiments, the transfer in step c) is started immediately after the transfer in step b) is completed. In some embodiments, the transfer in step c) is started between approximately 5 minutes and 60 minutes after the transfer in step b) is completed. In some embodiments, the transfer in step c) is started within approximately 5 minutes after the transfer in step b) is completed. In some embodiments, the transfer in step c) is started within approximately 10 minutes after the transfer in step b) is completed. In some embodiments, the transfer in step c) is started within approximately 15 minutes after the transfer in step b) is completed.In some embodiments, the transfer in step c) is started within approximately 20 minutes after the transfer in step b) is completed. In some embodiments, the transfer in step c) is started within approximately 30 minutes after the transfer in step b) is completed. In some embodiments, the transfer in step c) is started within approximately 45 minutes after the transfer in step b) is completed. In some embodiments, the transfer in step c) is started within approximately 60 minutes after the transfer in step b) is completed. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are completed in less than approximately 90 minutes, less than approximately 60 minutes, less than approximately 50 minutes, less than approximately 40 minutes, less than approximately 35 minutes, less than approximately 30 minutes, less than approximately 25 minutes, or less than approximately 20 minutes, respectively. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are completed in less than approximately 60 minutes, respectively. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 50 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 40 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 35 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 30 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 25 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 20 minutes. In some embodiments, the incubation in steps b) and c) is performed for a period of about 5 to about 20 minutes, about 10 to about 20 minutes, about 5 to about 15 minutes, about 10 to about 15 minutes, or about 15 to about 20 minutes.In some embodiments, incubation in steps b) and c) is performed between approximately 10 and 15 minutes. In some embodiments, incubation in steps b) and c) is performed between approximately 5 minutes, 10 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, or 20 minutes. In some embodiments, incubation in steps b) and c) is performed between approximately 10 minutes. In some embodiments, incubation in steps b) and c) is performed between approximately 12 minutes. In some embodiments, incubation in steps b) and c) is performed between approximately 15 minutes. In some embodiments, transfer in steps b) and c) is performed over a period of approximately 1 to 12 hours. In some embodiments, transfer in steps b) and c) is performed over a period of approximately 1 to 8 hours. In some embodiments, transfer in steps b) and c) is performed over a period of approximately 1 to 6 hours. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 1 hour to about 5 hours. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 1 hour to about 4 hours. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 1 hour to about 3 hours. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 12 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 9 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 8 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 7 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 6 hours or less.In some embodiments, the transfer in steps b) and c) is carried out over a period of about 5 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 5 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 3 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 2 hours or less. In some embodiments, the transfer in steps b) and c) is carried out simultaneously or sequentially in any order. In some embodiments, the transfer in steps b) and c) is carried out sequentially in any order. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are carried out in the same manner. In some embodiments, the cell culture is a suspension culture. In some embodiments, the cell culture contains HEK293 cells adapted for growth in suspension culture. In some embodiments, the cell culture has a volume between about 400 liters and about 5,000 liters. In some embodiments, the cell culture has a volume of approximately 500 liters. In some embodiments, the cell culture has a volume of approximately 2,000 liters. In some embodiments, one or more polynucleotides comprise one or more helper genes, rep genes, cap genes, and transgenes (e.g., target genes). In some embodiments, the translocation reagent comprises a cationic polymer. In some embodiments, the translocation reagent comprises PEI. In some embodiments, the recombinant virus particles are recombinant adeno-associated virus (rAAV) particles. In some embodiments, one or more polynucleotides comprise a mixture of three polynucleotides (one encoding cap and rep genes, one encoding adenovirus helper functions necessary for packaging (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and one encoding the rAAV genome to be packaged).In some embodiments, the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV particles have an AAV capsid protein of a serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles have an AAV capsid protein with high sequence homology to AAV8 or AAV9, for example, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0088] In some embodiments, the present disclosure provides a method for increasing the generation of recombinant virus particles, a) Prepare a suspension cell culture of approximately 200 liters to approximately 20,000 liters containing a population of cells capable of producing recombinant virus particles, b) To form a first mixture, one or more polynucleotides are mixed with at least one translocation reagent; the mixture is incubated to form a polynucleotide:translocation reagent complex; and the polynucleotide:translocation reagent complex is transferred to a suspension culture for translocation into cells. c) To form a second mixture, one or more polynucleotides are mixed with at least one translocation reagent; the mixture is incubated to form a polynucleotide:translocation reagent complex; and the polynucleotide:translocation reagent complex is transferred to a suspension culture for translocation into cells. d) Maintain the cell culture containing transfected cells under conditions that enable the generation of recombinant virus particles. Includes, A method is provided in which one or more polynucleotides contain genes necessary for the generation of recombinant viral particles, and step c) is repeated one or more times. In some embodiments, the method produces at least about twice as many rAAV particles, measured as GC / ml, compared to a reference method comprising a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfer reagent complex. In some embodiments, the method increases rAAV production by at least about 50%, at least about 75%, or at least about 100% compared to a reference method comprising a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfer reagent complex. In some embodiments, the method disclosed herein increases rAAV production by at least about 2, at least about 3, or at least about 5 times compared to a reference method comprising a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfer reagent complex. In some embodiments, step c) is repeated one more time. In some embodiments, step c) is repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, step c) is repeated 2 times. In some embodiments, step c) is repeated 3 times. In some embodiments, step c) is repeated 4 times. In some embodiments, step c) is repeated 5 times. In some embodiments, step c) is repeated 6 times. In some embodiments, step c) is repeated 7 times. In some embodiments, step c) is repeated 8 times. In some embodiments, step c) is repeated 7 times. In some embodiments, step c) is repeated 9 times. In some embodiments, step c) is repeated 7 times. In some embodiments, step c) is repeated 10 times. In some embodiments, the combined volume of the polynucleotide:transfer reagent complex transferred to the suspension culture is between approximately 5% and approximately 20% of the volume of the cell culture in step a).In some embodiments, the combined volume of the polynucleotide:transfer reagent complex transferred to the suspension culture is between approximately 7% and 15% of the volume of the cell culture in step a). In some embodiments, the combined volume of the polynucleotide:transfer reagent complex transferred to the suspension culture is between approximately 10% of the volume of the cell culture in step a). In some embodiments, the transfer in step c) is started before the transfer in the preceding step is completed. In some embodiments, the transfer in step c) is started immediately after the transfer in the preceding step is completed. In some embodiments, the transfer in step c) is started between approximately 5 and 60 minutes after the transfer in the preceding step is completed. In some embodiments, the transfer in step c) is started within approximately 5 minutes after the transfer in the preceding step is completed. In some embodiments, the transfer in step c) is started within approximately 10 minutes after the transfer in the preceding step is completed. In some embodiments, the transfer in step c) is started within approximately 15 minutes after the transfer in the preceding step is completed. In some embodiments, the transfer in step c) is initiated within approximately 20 minutes after the completion of the transfer in the preceding step. In some embodiments, the transfer in step c) is initiated within approximately 30 minutes after the completion of the transfer in the preceding step. In some embodiments, the transfer in step c) is initiated within approximately 45 minutes after the completion of the transfer in the preceding step. In some embodiments, the transfer in step c) is initiated within approximately 60 minutes after the completion of the transfer in the preceding step. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are completed in less than approximately 90 minutes, less than approximately 60 minutes, less than approximately 50 minutes, less than approximately 40 minutes, less than approximately 35 minutes, less than approximately 30 minutes, less than approximately 25 minutes, or less than approximately 20 minutes, respectively. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are completed in less than approximately 60 minutes, respectively.In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 50 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 40 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 35 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 30 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 25 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 20 minutes. In some embodiments, incubation in steps b) and c) is performed for about 5 to about 20 minutes, about 10 to about 20 minutes, about 5 to about 15 minutes, about 10 to about 15 minutes, or about 15 to about 20 minutes. In some embodiments, incubation in steps b) and c) is performed for about 10 to about 15 minutes. In some embodiments, incubation in steps b) and c) is performed for about 5 minutes, about 10 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, or about 20 minutes. In some embodiments, incubation in steps b) and c) is performed for about 10 minutes. In some embodiments, incubation in steps b) and c) is performed for about 12 minutes. In some embodiments, incubation in steps b) and c) is performed for about 15 minutes. In some embodiments, the transfer of steps b) and c) is carried out over a period of time ranging from about 1 hour to about 12 hours. In some embodiments, the transfer of steps b) and c) is carried out over a period of time ranging from about 1 hour to about 8 hours.In some embodiments, the transfer in steps b) and c) is carried out over a period of about 1 hour to about 6 hours. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 1 hour to about 5 hours. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 1 hour to about 4 hours. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 1 hour to about 3 hours. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 12 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 9 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 8 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 7 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 6 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 5 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 5 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 3 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 2 hours or less. In some embodiments, the transfer in steps b) and c) is carried out simultaneously or sequentially in any order. In some embodiments, the transfer in steps b) and c) is carried out sequentially in any order. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are carried out in the same manner. In some embodiments, the cell culture is a suspension culture. In some embodiments, the cell culture contains HEK293 cells adapted for growth in suspension culture.In some embodiments, the cell culture has a volume between about 400 liters and about 5,000 liters. In some embodiments, the cell culture has a volume of about 500 liters. In some embodiments, the cell culture has a volume of about 2,000 liters. In some embodiments, the translocation reagent comprises a cationic polymer. In some embodiments, the translocation reagent comprises PEI. In some embodiments, the recombinant virus particles are recombinant adeno-associated virus (rAAV) particles. In some embodiments, one or more polynucleotides comprise one or more helper genes, rep genes, cap genes, and transgenes (e.g., the target gene or the rAAV genome to be packaged). In some embodiments, one or more polynucleotides comprise a mixture of three polynucleotides (one encoding the cap and rep genes, one encoding the adenovirus helper function required for packaging (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and one encoding the rAAV genome to be packaged). In some embodiments, the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV particles have an AAV capsid protein of a serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles have an AAV capsid protein with high sequence homology to AAV8 or AAV9, for example, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0089] In some embodiments, the present disclosure relates to a method for generating recombinant adeno-associated virus (rAAV) particles, a) Prepare a suspension cell culture of approximately 200 liters to approximately 20,000 liters containing a population of cells capable of producing rAAV, b) To form a first mixture, one or more polynucleotides are mixed with at least one translocation reagent; the mixture is incubated to form a polynucleotide:translocation reagent complex; and the polynucleotide:translocation reagent complex is transferred to a suspension cell culture for translocation into cells. c) To form a second mixture, one or more polynucleotides are mixed with at least one translocation reagent; the mixture is incubated to form a polynucleotide:translocation reagent complex; and the polynucleotide:translocation reagent complex is transferred to a suspension cell culture for translocation into cells. d) Maintain the suspension cell culture containing transfused cells under conditions that enable the generation of rAAV particles. Includes, The present disclosure provides a method in which a translocation reagent comprises PEI and one or more polynucleotides comprising genes necessary for the production of rAAV particles. In some embodiments, the present disclosure provides a method for increasing the production of recombinant adeno-associated virus (rAAV) particles, a) Prepare a suspension cell culture of approximately 200 liters to approximately 20,000 liters containing a population of cells capable of producing rAAV particles, b) To form a first mixture, one or more polynucleotides are mixed with at least one translocation reagent; the mixture is incubated to form a polynucleotide:translocation reagent complex; and the polynucleotide:translocation reagent complex is transferred to a suspension culture for translocation into cells. c) To form a second mixture, one or more polynucleotides are mixed with at least one translocation reagent; the mixture is incubated to form a polynucleotide:translocation reagent complex; and the polynucleotide:translocation reagent complex is transferred to a suspension culture for translocation into cells. d) A method is provided comprising maintaining a suspension cell culture containing transfused cells under conditions that enable the generation of rAAV particles, wherein the transfusion reagent comprises PEI and one or more polynucleotides comprises genes necessary for the generation of rAAV particles. In some embodiments, the method generates at least about twice as many rAAV particles, as measured as GC / ml, compared to a reference method comprising a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfusion reagent complex. In some embodiments, the method increases rAAV generation by at least about 50%, at least about 75%, or at least about 100% compared to a reference method comprising a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfusion reagent complex. In some embodiments, the method disclosed herein increases rAAV generation by at least about 2, at least about 3, or at least about 5 times compared to a reference method comprising a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfusion reagent complex. In some embodiments, the Disclosure provides a method for generating recombinant adeno-associated virus (rAAV) particles, comprising: a) preparing a suspension cell culture in a volume between about 200 liters and about 20,000 liters containing a population of cells capable of generating rAAV particles; b) mixing one or more polynucleotides with at least one translocation reagent to form a first mixture, and incubating the mixture to form a polynucleotide:translocation reagent complex; c) mixing one or more polynucleotides with at least one translocation reagent to form a second mixture, and incubating the mixture to form a polynucleotide:translocation reagent complex, and transferring the polynucleotide:translocation reagent complex to the culture for translocation into cells; and d) maintaining the suspension cell culture containing the translocated cells under conditions that enable the generation of rAAV particles, wherein the translocation reagent contains PEI, one or more polynucleotides contain genes necessary for the generation of rAAV particles, and step c) is repeated one or more times. In some embodiments, step c) is repeated one more time.In some embodiments, step c) is repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, step c) is repeated 2 times. In some embodiments, step c) is repeated 3 times. In some embodiments, step c) is repeated 4 times. In some embodiments, step c) is repeated 5 times. In some embodiments, step c) is repeated 6 times. In some embodiments, step c) is repeated 7 times. In some embodiments, step c) is repeated 8 times. In some embodiments, step c) is repeated 7 times. In some embodiments, step c) is repeated 9 times. In some embodiments, step c) is repeated 7 times. In some embodiments, step c) is repeated 10 times. In some embodiments, the Disclosure provides a method for increasing the production of recombinant adeno-associated virus (rAAV) particles, comprising: a) preparing a suspension cell culture in a volume between about 200 liters and about 20,000 liters containing a population of cells capable of producing rAAV particles; b) mixing one or more polynucleotides with at least one translocation reagent to form a first mixture, and incubating the mixture to form a polynucleotide:translocation reagent complex; c) mixing one or more polynucleotides with at least one translocation reagent to form a second mixture, and incubating the mixture to form a polynucleotide:translocation reagent complex, and transferring the polynucleotide:translocation reagent complex to the culture for translocation into cells; and d) maintaining the suspension cell culture containing the translocated cells under conditions that enable the production of rAAV particles, wherein the translocation reagent contains PEI, one or more polynucleotides contain genes necessary for the production of rAAV particles, and step c) is repeated one or more times. In some embodiments, the method produces at least about twice as many rAAV particles, measured as GC / ml, compared to a reference method that involves a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfer reagent complexes.In some embodiments, the method increases rAAV production by at least about 50%, at least about 75%, or at least about 100% compared to a reference method comprising a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfer reagent complex. In some embodiments, the method disclosed herein increases rAAV production by at least about 2 times, at least about 3 times, or at least about 5 times compared to a reference method comprising a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfer reagent complex. In some embodiments, the combined volume of polynucleotide:transfer reagent complex transferred to the suspension cell culture is between about 5% and about 20% of the volume of the suspension cell culture in step a). In some embodiments, the combined volume of the polynucleotide:transfer reagent complex transferred to the suspension cell culture is between approximately 7% and 15% of the volume of the suspension cell culture in step a). In some embodiments, the combined volume of the polynucleotide:transfer reagent complex transferred to the suspension cell culture is between approximately 10% of the volume of the suspension cell culture in step a). In some embodiments, the transfer in step c) is started before the transfer in the preceding step is completed. In some embodiments, the transfer in step c) is started immediately after the transfer in the preceding step is completed. In some embodiments, the transfer in step c) is started between approximately 5 and 60 minutes after the transfer in the preceding step is completed. In some embodiments, the transfer in step c) is started within approximately 5 minutes after the transfer in the preceding step is completed. In some embodiments, the transfer in step c) is started within approximately 10 minutes after the transfer in the preceding step is completed. In some embodiments, the transfer in step c) is started within approximately 15 minutes after the transfer in the preceding step is completed. In some embodiments, the transfer in step c) is initiated within approximately 20 minutes after the completion of the transfer in the preceding step.In some embodiments, the transfer in step c) is started within approximately 30 minutes after the transfer in the preceding step is completed. In some embodiments, the transfer in step c) is started within approximately 45 minutes after the transfer in the preceding step is completed. In some embodiments, the transfer in step c) is started within approximately 60 minutes after the transfer in step b) is completed. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are completed in less than approximately 90 minutes, less than approximately 60 minutes, less than approximately 50 minutes, less than approximately 40 minutes, less than approximately 35 minutes, less than approximately 30 minutes, less than approximately 25 minutes, or less than approximately 20 minutes, respectively. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are completed in less than approximately 60 minutes, respectively. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are completed in less than approximately 50 minutes, respectively. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 40 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 35 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 30 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 25 minutes. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are each completed in less than approximately 20 minutes. In some embodiments, the incubation in steps b) and c) is performed for about 5 to about 20 minutes, about 10 to about 20 minutes, about 5 to about 15 minutes, about 10 to about 15 minutes, or about 15 to about 20 minutes. In some embodiments, the incubation in steps b) and c) is performed for about 10 to about 15 minutes.In some embodiments, incubation in steps b) and c) is performed for about 5 minutes, about 10 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, or about 20 minutes. In some embodiments, incubation in steps b) and c) is performed for about 10 minutes. In some embodiments, incubation in steps b) and c) is performed for about 12 minutes. In some embodiments, incubation in steps b) and c) is performed for about 15 minutes. In some embodiments, transfer in steps b) and c) is performed over a period of about 1 to about 12 hours. In some embodiments, transfer in steps b) and c) is performed over a period of about 1 to about 8 hours. In some embodiments, transfer in steps b) and c) is performed over a period of about 1 to about 6 hours. In some embodiments, transfer in steps b) and c) is performed over a period of about 1 to about 5 hours. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 1 to 4 hours. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 1 to 3 hours. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 12 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 9 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 8 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 7 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 6 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 5 hours or less. In some embodiments, the transfer in steps b) and c) is carried out over a period of about 5 hours or less.In some embodiments, the transfers in steps b) and c) are carried out over a period of approximately 3 hours or less. In some embodiments, the transfers in steps b) and c) are carried out over a period of approximately 2 hours or less. In some embodiments, the transfers in steps b) and c) are carried out simultaneously or sequentially in any order. In some embodiments, the transfer in steps b) and c) is carried out sequentially in any order. In some embodiments, the mixing, incubation, and transfer in steps b) and c) are carried out in the same manner. In some embodiments, the suspension cell culture contains HEK293 cells adapted for growth in suspension cell culture. In some embodiments, the suspension cell culture has a volume between about 400 liters and about 10,000 liters. In some embodiments, the cell culture has a volume of about 500 liters. In some embodiments, the cell culture has a volume of about 2,000 liters. In some embodiments, one or more polynucleotides contain one or more helper genes, rep genes, cap genes, and transgenes (e.g., the target gene or the rAAV genome to be packaged). In some embodiments, one or more polynucleotides comprise a mixture of three polynucleotides (one encoding cap and rep genes, one encoding adenovirus helper functions necessary for packaging (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and one encoding the rAAV genome to be packaged). In some embodiments, the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV particles have an AAV capsid protein of a serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles have AAV capsid proteins with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0090] Recombinant virus particle production systems based on translocation are known to those skilled in the art. See, for example, Reiser et al., Gene Ther 7(11):910-3(2000); Dull et al., J Virol. 72(11):8463-8471(1998); Hoffmann et al., PNAS 97(11)6108-6113(2000); Milian et al., Vaccine 35(26):3423-3430 (2017) (each of these is incorporated herein by reference in whole). The methods disclosed herein can be used to produce recombinant virus particles in translocation-based production systems. In some embodiments, the recombinant virus particles are recombinant dengue virus, recombinant Ebola virus, recombinant human papillomavirus (HPV), recombinant human immunodeficiency virus (HIV), recombinant adeno-associated virus (AAV), recombinant lentivirus, recombinant influenza virus, recombinant varicella-stomatitis virus (VSV), recombinant poliovirus, recombinant adenovirus, recombinant retrovirus, recombinant vaccinia, recombinant reovirus, recombinant measles virus, recombinant Newcastle disease virus (NDV), recombinant herpes zoster virus (HZV), recombinant herpes simplex virus (HSV), or recombinant baculovirus. In some embodiments, the recombinant virus particles are recombinant adeno-associated virus (AAV), recombinant lentivirus, or recombinant influenza virus. In some embodiments, the recombinant virus particles are recombinant lentivirus. In some embodiments, the recombinant virus particles are recombinant influenza virus. In some embodiments, the recombinant virus particles are recombinant baculovirus. In some embodiments, the recombinant virus particles are recombinant adeno-associated virus (AAV). In some embodiments, the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV particles have an AAV capsid protein of a serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8.In some embodiments, the rAAV particles have AAV capsid proteins with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0091] Any suitable translocation reagent known in the art for translocation into cells can be used to generate recombinant viral particles (e.g., rAAV particles) according to the methods disclosed herein. In some embodiments, the cells are HEK293 cells (e.g., HEK293 cells adapted for suspension culture). In some embodiments, the methods disclosed herein include translocation into cells using chemically based translocation methods. In some embodiments, the methods disclosed herein include translocation into cells using cationic organic carriers. See, for example, Gigante et al., MedChemComm 10(10):1692-1718(2019); Damen et al., MedChemComm 9(9):1404-1425(2018) (each of these is incorporated herein by reference in whole). In some embodiments, the cationic organic carrier includes lipids, e.g., DOTMA, DOTAP, helper lipids (Dope, cholesterol), and combinations thereof. In some embodiments, the cationic organic carrier comprises polyvalent cationic lipids, such as DOSPA, DOGS, and mixtures thereof. In some embodiments, the cationic organic carrier comprises bipolar lipids, or bora amphiphiles (bola). In some embodiments, the cationic organic carrier comprises biologically reducing and / or dimerizable lipids. In some embodiments, the cationic organic carrier comprises gemin surfactants. In some embodiments, the cationic organic carrier comprises Lipofectin®, Transfectam®, Lipofectamine®, Lipofectamine 2000®, or Lipofectamin PLUS 2000®.In some embodiments, the cationic organic carrier includes polymers, such as poly(L-lysine) (PLL), polyethyleneimine (PEI), polysaccharides (chitosan, dextran, cyclodextrin (CD)), poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA), and dendrimers (polyamidoamine (PAMAM), poly(propyleneimine) (PPI)). In some embodiments, the cationic organic carrier includes peptides, such as peptides rich in basic amino acids (CWL). 18 The reagents include a cell-permeable peptide (CPP) (Arg-rich peptide (octaarginine, TAT)), a nuclear localization signal (NLS) (SV40), and a targeting agent (RGD). In some embodiments, the cationic organic carrier comprises a polymer (e.g., PEI) combined with a cationic liposome. Paris et al., Molecules 25(14):3277 (2020) (the whole of which is incorporated herein by reference). In some embodiments, the translocation reagent comprises calcium phosphate, a highly branched organic compound (dendrimer), a cationic polymer (e.g., DEAE dextran or polyethyleneimine (PEI)), and lipofection.

[0092] In some embodiments, the translocation reagent comprises poly(L-lysine) (PLL), polyethyleneimine (PEI), linear PEI, branched PEI, dextran, cyclodextrin (CD), poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA), polyamidoamine (PAMAM), poly(propyleneimine) (PPI), or mixtures thereof. In some embodiments, the translocation reagent comprises polyethyleneimine (PEI), linear PEI, branched PEI, or mixtures thereof. In some embodiments, the translocation reagent comprises polyethyleneimine (PEI). In some embodiments, the translocation reagent comprises linear PEI. In some embodiments, the translocation reagent comprises branched PEI. In some embodiments, the translocation reagent comprises polyethyleneimine (PEI) having a molecular weight between about 5 and about 25 kDa. In some embodiments, the translocation reagent comprises PEGylated polyethyleneimine (PEI). In some embodiments, the translocation reagent comprises modified polyethyleneimine (PEI) to which hydrophobic moieties (e.g., cholesterol, choline, alkyl groups, and several amino acids) are attached.

[0093] The composition polynucleotide:transfer reagent complex can be prepared by any method known to those skilled in the art. In some embodiments, mixing one or more polynucleotides with at least one transfer reagent involves diluting each of the transfer reagent and one or more polynucleotides in a sterile liquid (e.g., tissue culture medium) and mixing the diluted transfer reagent and the diluted one or more polynucleotides. In some embodiments, mixing involves transferring the diluted one or more polynucleotides and the diluted at least one transfer reagent from two separate containers to a new container. In some embodiments, transferring the diluted one or more polynucleotides and the diluted at least one transfer reagent to a new container is carried out at a rate of about 500 ml / min, about 1 liter / min, about 2 liters / min, about 3 liters / min, about 4 liters / min, about 5 liters / min, about 6 liters / min, about 7 liters / min, about 8 liters / min, about 9 liters / min, or about 10 liters / min. In some embodiments, the transfer is carried out at a rate of about 3 liters / min. In some embodiments, the transfer is carried out at a rate of approximately 4 liters / minute. In some embodiments, the transfer is carried out at a rate of approximately 5 liters / minute. In some embodiments, the transfer is carried out at a rate of approximately 6 liters / minute. In some embodiments, the mixing of one or more diluted polynucleotides with at least one diluted translocation reagent is carried out by an in-line mixer. In some embodiments, the in-line mixer is a low-shear in-line mixer. In some embodiments, the in-line mixer is a static in-line mixer. In-line mixers suitable for mixing polynucleotides and translocation reagents are known in the art and can be obtained, for example, from Sartorius (Flexsafe® Pro Mixer), Analytical Scientific Instruments US, Inc (HYPERSHEAR®), Fluitec, or STRIKO.Those skilled in the art will understand that dilution and mixing are performed to produce a composition containing the translocation reagent and the polynucleotide in desired ratios and concentrations. In some embodiments, dilution and mixing of at least one translocation reagent and one or more polynucleotides produce a composition containing the translocation reagent and the polynucleotide in a weight ratio between about 1:5 and 5:1. In some embodiments, the weight ratio of the translocation reagent and the polynucleotide is between about 1:3 and 3:1. In some embodiments, the weight ratio of the translocation reagent and the polynucleotide is between about 1:3 and 1:1. In some embodiments, the weight ratio of the translocation reagent and the polynucleotide is between about 1:2 and 1:1.5. In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is approximately 1:5, 1:4, 1:3, 1:2.5, 1:2, 1:1.75, 1:1.5, 1:1.25, 1:1, 1.25:1, 1.5:1, 1.75:1, 2:1, 2.5:1, 3:1, 4:1, or 5:1. In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is approximately 1:2. In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is approximately 1:1.75. In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is approximately 1:1.5. In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is approximately 1:1.25. In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is approximately 1:1. In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is approximately 1.25:1. In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is approximately 1.5:1. In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is approximately 1.75:1. In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is approximately 2:1. In some embodiments, the composition contains one or more polynucleotides between approximately 1 μg and approximately 20 μg. In some embodiments, the one or more polynucleotides contain three plasmids. In some embodiments, the one or more polynucleotides contain two plasmids.In some embodiments, one or more polynucleotides comprise one plasmid. In some embodiments, the recombinant virus is recombinant AAV, and one or more polynucleotides comprise a mixture of three polynucleotides (one encoding cap and rep genes, one encoding adenovirus helper functions necessary for packaging (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and one encoding the rAAV genome to be packaged). In some embodiments, the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV particles have an AAV capsid protein of a serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles have AAV capsid proteins with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the translocation reagent is PEI.

[0094] In some embodiments, a composition comprising a translocation reagent and one or more polynucleotides is incubated to allow the formation of a polynucleotide:translocation reagent complex. In some embodiments, incubation is performed at room temperature. In some embodiments, incubation includes shaking the composition, for example, in a shaker between about 100 rpm and about 200 rpm. In some embodiments, incubation is performed for about 5 to about 20 minutes, about 10 to about 20 minutes, about 5 to about 15 minutes, about 10 to about 15 minutes, or about 15 to about 20 minutes. In some embodiments, incubation is performed between about 5 to about 20 minutes. In some embodiments, incubation is performed for about 10 to about 15 minutes. In some embodiments, incubation is performed for about 5 minutes, about 10 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, or about 20 minutes. In some embodiments, incubation is performed for about 11 minutes. In some embodiments, incubation is performed for about 12 minutes. In some embodiments, incubation is performed for about 13 minutes. In some embodiments, incubation is performed for about 14 minutes. In some embodiments, incubation is performed for about 15 minutes. In some embodiments, incubation is performed for 15 minutes or less. In some embodiments, incubation is performed for 10 minutes or less. In some embodiments, incubation is performed for about 5 minutes, about 10 minutes, or about 15 minutes. In some embodiments, incubation is performed for about 10 minutes. In some embodiments, the length of incubation is such that mixing, incubation, and transfer can be completed in less than about 90 minutes, less than about 60 minutes, less than about 50 minutes, less than about 40 minutes, less than about 35 minutes, less than about 30 minutes, less than about 25 minutes, or less than about 20 minutes. In some embodiments, mixing, incubation, and transfer can be completed in less than about 30 minutes. In some embodiments, one or more polynucleotides contain genes necessary for the production of recombinant AAV particles.In some embodiments, the translocation reagent includes PEI.

[0095] Methods for transferring polynucleotide:transfer reagent complexes to suspension cultures are known to those skilled in the art. In some embodiments, the transfer is carried out using a peristaltic pump. In some embodiments, the transfer is carried out at a rate between about 100 ml / min and about 10 liters / min. In some embodiments, the transfer is carried out at a rate of about 500 ml / min, about 1 liter / min, about 2 liters / min, about 3 liters / min, about 4 liters / min, about 5 liters / min, about 6 liters / min, about 7 liters / min, about 8 liters / min, about 9 liters / min, or about 10 liters / min. In some embodiments, the transfer is carried out at a rate of about 3 liters / min. In some embodiments, the transfer is carried out at a rate of about 4 liters / min. In some embodiments, the transfer is carried out at a rate of about 5 liters / min. In some embodiments, the transfer is carried out at a rate of about 6 liters / min. In some embodiments, the transfer is carried out at a rate of about 7 liters / min. In some embodiments, the transfer is carried out at a rate of about 8 liters / minute. In some embodiments, the transfer is carried out at a rate of about 9 liters / minute. In some embodiments, the transfer is carried out at a rate of about 10 liters / minute. In some embodiments, the transfer rate is set so that mixing, incubation, and transfer are completed in less than about 90 minutes, less than about 60 minutes, less than about 50 minutes, less than about 40 minutes, less than about 35 minutes, less than about 30 minutes, less than about 25 minutes, or less than about 20 minutes. In some embodiments, mixing, incubation, and transfer are completed in less than about 30 minutes. In some embodiments, mixing, incubation, and transfer are completed in less than about 35 minutes. In some embodiments, the polynucleotide contains genes necessary for the production of recombinant AAV particles. In some embodiments, the translocation reagent contains PEI. In some embodiments, the culture contains HEK293 cells (e.g., HEK293 cells adapted for suspension culture).

[0096] In some embodiments, separately generated polynucleotide:transfer reagent complexes are produced using the same process. In some embodiments, the same process is used to separately mix one or more polynucleotides with at least one transfer reagent to form a first mixture, to incubate the mixture to form a polynucleotide:transfer reagent complex, and to transfer the polynucleotide:transfer reagent complex to a suspension culture for transfusion into cells. In some embodiments, separately transferring the polynucleotide:transfer reagent complex to a suspension culture includes transferring the same volume of polynucleotide:transfer reagent complex. In some embodiments, separately transferring the polynucleotide:transfer reagent complex to a suspension culture includes transferring different volumes of polynucleotide:transfer reagent complex. In some embodiments, one or more polynucleotides include genes necessary for the production of recombinant AAV particles. In some embodiments, the transfer reagent includes PEI. In some embodiments, the culture includes HEK293 cells (e.g., HEK293 cells adapted for suspension culture).

[0097] In some embodiments, the combined volume of the polynucleotide:transfer reagent complex transferred to the cell culture is between approximately 5% and approximately 20% of the volume of the cell culture containing a population of cells capable of producing recombinant viral particles (e.g., rAAV particles). In some embodiments, the combined volume of the transferred polynucleotide:transfer reagent complex is between approximately 7% and approximately 15% of the volume of the cell culture. In some embodiments, the combined volume of the transferred polynucleotide:transfer reagent complex is approximately 10% of the volume of the cell culture. In some embodiments, one or more polynucleotides contain genes necessary for the production of recombinant AAV particles. In some embodiments, the transfer reagent contains PEI. In some embodiments, the culture contains HEK293 cells (e.g., HEK293 cells adapted for suspension culture).

[0098] In some embodiments, the combined volume of the polynucleotide:transfer reagent complex transferred to the cell culture ranges from approximately 0.1 μg of one or more polynucleotides / 10E+6 live cells / ml to approximately 5 μg of one or more polynucleotides / 10E+6 live cells / ml. In some embodiments, the combined volume of the polynucleotide:transfer reagent complex transferred to the cell culture ranges from approximately 0.2 μg of one or more polynucleotides / 10E+6 live cells / ml to approximately 2 μg of one or more polynucleotides / 10E+6 live cells / ml. In some embodiments, the combined volume of the polynucleotide:transfer reagent complex transferred to the cell culture ranges from approximately 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 μg of one or more polynucleotides / 10E+6 live cells / ml. In some embodiments, one or more polynucleotides contain genes necessary for the production of recombinant AAV particles. In some embodiments, the transfer reagent contains PEI. In some embodiments, the culture includes HEK293 cells (e.g., HEK293 cells adapted for suspension culture).

[0099] In some embodiments, the cell culture has a volume between approximately 400 liters and approximately 20,000 liters. In some embodiments, the cell culture has a volume between approximately 500 liters and approximately 20,000 liters. In some embodiments, the cell culture has a volume between approximately 700 liters and approximately 20,000 liters. In some embodiments, the cell culture has a volume between approximately 1,000 liters and approximately 20,000 liters. In some embodiments, the cell culture has a volume between approximately 400 liters and approximately 10,000 liters. In some embodiments, the cell culture has a volume between approximately 500 liters and approximately 10,000 liters. In some embodiments, the cell culture has a volume between approximately 700 liters and approximately 10,000 liters. In some embodiments, the cell culture has a volume between approximately 1,000 liters and approximately 10,000 liters. In some embodiments, the cell culture has a volume between approximately 400 liters and approximately 5,000 liters. In some embodiments, the cell culture has a volume between approximately 500 liters and approximately 5,000 liters. In some embodiments, the cell culture has a volume between approximately 700 liters and approximately 5,000 liters. In some embodiments, the cell culture has a volume between approximately 1,000 liters and approximately 5,000 liters. In some embodiments, the cell culture volume referred to herein is the capacity of the final bioreactor / vessel as shown in Table 1. In some embodiments, the culture contains HEK293 cells (e.g., HEK293 cells adapted for suspension culture).

[0100] In some embodiments, the cell culture has a volume between approximately 200 liters and approximately 5,000 liters. In some embodiments, the cell culture has a volume between approximately 200 liters and approximately 2,000 liters. In some embodiments, the cell culture has a volume between approximately 200 liters and approximately 1,000 liters. In some embodiments, the cell culture has a volume between approximately 200 liters and approximately 500 liters. In some embodiments, the cell culture volume referred to herein is the capacity of the final bioreactor / vessel as shown in Table 1. In some embodiments, the culture contains HEK293 cells (e.g., HEK293 cells adapted for suspension culture).

[0101] In some embodiments, the cell culture has a volume of approximately 200 liters. In some embodiments, the cell culture has a volume of approximately 300 liters. In some embodiments, the cell culture has a volume of approximately 400 liters. In some embodiments, the cell culture has a volume of approximately 500 liters. In some embodiments, the cell culture has a volume of approximately 750 liters. In some embodiments, the cell culture has a volume of approximately 1,000 liters. In some embodiments, the cell culture has a volume of approximately 2,000 liters. In some embodiments, the cell culture has a volume of approximately 3,000 liters. In some embodiments, the cell culture has a volume of approximately 5,000 liters. In some embodiments, the culture contains HEK293 cells (e.g., HEK293 cells adapted for suspension culture).

[0102] In some embodiments, the method described herein includes transferring two separately produced polynucleotide:transfer reagent complexes in volumes of approximately 20 liters (e.g., approximately 21 liters) to approximately 400 liters of cell culture. In some embodiments, the method described herein includes transferring three separately produced polynucleotide:transfer reagent complexes in volumes of approximately 20 liters to approximately 600 liters of cell culture. In some embodiments, the method described herein includes transferring four separately produced polynucleotide:transfer reagent complexes in volumes of approximately 20 liters to approximately 800 liters of cell culture. In some embodiments, the method described herein includes transferring five separately produced polynucleotide:transfer reagent complexes in volumes of approximately 20 liters to approximately 1,000 liters of cell culture. In some embodiments, the method described herein includes transferring six separately produced polynucleotide:transfer reagent complexes in volumes of approximately 20 liters to approximately 1,200 liters of cell culture. In some embodiments, the method described herein includes transferring seven separately produced polynucleotide:transfer reagent complexes in volumes of approximately 20 liters each to approximately 1,400 liters of cell culture. In some embodiments, the method described herein includes transferring eight separately produced polynucleotide:transfer reagent complexes in volumes of approximately 20 liters each to approximately 1,600 liters of cell culture. In some embodiments, the method described herein includes transferring nine separately produced polynucleotide:transfer reagent complexes in volumes of approximately 20 liters each to approximately 1,800 liters of cell culture. In some embodiments, the method described herein includes transferring ten separately produced polynucleotide:transfer reagent complexes in volumes of approximately 20 liters each to approximately 2,000 liters of cell culture. For reference, non-limiting examples of the volumes of separately produced transfer reagent complex mixtures are shown in Table 1.

[0103] In some embodiments, the method described herein involves transferring one polynucleotide:transfer reagent complex in a volume of about 40 liters (e.g., about 42 liters) to about 400 liters of cell culture. In some embodiments, the method described herein involves transferring two separately produced polynucleotide:transfer reagent complexes in volumes of about 40 liters each to about 800 liters of cell culture. In some embodiments, the method described herein involves transferring four separately produced polynucleotide:transfer reagent complexes in volumes of about 40 liters each to about 1,600 liters of cell culture. For reference, Table 1 shows non-limiting examples of the volumes of separately produced transfer reagent complex mixtures.

[0104] [Table 1]

[0105] In some embodiments, the transfer rate is set so that mixing, incubation, and transfer are completed in less than about 90 minutes, less than about 60 minutes, less than about 50 minutes, less than about 40 minutes, less than about 35 minutes, less than about 30 minutes, less than about 25 minutes, or less than about 20 minutes. In some embodiments, mixing, incubation, and transfer are completed in less than about 30 minutes. In some embodiments, one or more polynucleotides contain genes necessary for the production of recombinant AAV particles. In some embodiments, the translocation reagent contains PEI. In some embodiments, one or more polynucleotides contain one or more helper genes, rep genes, cap genes, and transgenes (e.g., the target gene or rAAV genome to be packaged). In some embodiments, the culture contains HEK293 cells (e.g., HEK293 cells adapted for suspension culture).

[0106] In some embodiments, the method described herein includes transferring two separately produced polynucleotide:transfer reagent complexes in volumes of approximately 30 liters each to about 600 liters of cell culture. In some embodiments, the method described herein includes transferring three separately produced polynucleotide:transfer reagent complexes in volumes of approximately 30 liters each to about 900 liters of cell culture. In some embodiments, the method described herein includes transferring four separately produced polynucleotide:transfer reagent complexes in volumes of approximately 30 liters each to about 1,200 liters of cell culture. In some embodiments, the method described herein includes transferring five separately produced polynucleotide:transfer reagent complexes in volumes of approximately 30 liters each to about 1,500 liters of cell culture. In some embodiments, the method described herein includes transferring six separately produced polynucleotide:transfer reagent complexes in volumes of approximately 30 liters each to about 1,800 liters of cell culture. In some embodiments, the method described herein includes transferring seven separately produced polynucleotide:transfer reagent complexes in approximately 30 liters in volume to approximately 2,100 liters of cell culture. In some embodiments, the method described herein includes transferring eight separately produced polynucleotide:transfer reagent complexes in approximately 30 liters in volume to approximately 2,400 liters of cell culture. In some embodiments, the method described herein includes transferring nine separately produced polynucleotide:transfer reagent complexes in approximately 30 liters in volume to approximately 2,700 liters of cell culture. In some embodiments, the method described herein includes transferring ten separately produced polynucleotide:transfer reagent complexes in approximately 30 liters in volume to approximately 3,000 liters of cell culture. In some embodiments, the transfer rate is set so that mixing, incubation, and transfer are completed in less than approximately 90 minutes, less than approximately 60 minutes, less than approximately 50 minutes, less than approximately 40 minutes, less than approximately 35 minutes, less than approximately 30 minutes, less than approximately 25 minutes, or less than approximately 20 minutes. In some embodiments, mixing, incubation, and transfer are completed in less than approximately 30 minutes.In some embodiments, one or more polynucleotides contain genes necessary for the generation of recombinant AAV particles. In some embodiments, the translocation reagent contains PEI. In some embodiments, the culture contains HEK293 cells (e.g., HEK293 cells adapted for suspension culture).

[0107] In some embodiments, mixing, incubation, and transfer are completed in less than approximately 90 minutes, less than approximately 60 minutes, less than approximately 50 minutes, less than approximately 40 minutes, less than approximately 35 minutes, less than approximately 30 minutes, less than approximately 25 minutes, or less than approximately 20 minutes. In some embodiments, mixing, incubation, and transfer are completed in less than approximately 60 minutes. In some embodiments, mixing, incubation, and transfer are completed in less than approximately 50 minutes. In some embodiments, mixing, incubation, and transfer are completed in less than approximately 40 minutes. In some embodiments, mixing, incubation, and transfer are completed in less than approximately 35 minutes. In some embodiments, mixing, incubation, and transfer are completed in less than approximately 30 minutes. In some embodiments, mixing, incubation, and transfer are completed in less than approximately 25 minutes. In some embodiments, mixing, incubation, and transfer are completed in less than approximately 20 minutes.

[0108] The transfer of separately generated polynucleotide:transfer reagent complexes to a cell culture can be carried out simultaneously or sequentially. Simultaneous transfer includes overlapping transfers. In some embodiments, separately generated polynucleotide:transfer reagent complexes are transferred to the cell culture simultaneously. In some embodiments, separately generated polynucleotide:transfer reagent complexes are transferred to the cell culture sequentially. In some embodiments, the transfer of a volume of separately generated polynucleotide:transfer reagent complexes is initiated before the transfer of the preceding separately generated volume is completed. In some embodiments, the transfer of a volume of separately generated polynucleotide:transfer reagent complexes is initiated immediately after the transfer of the preceding separately generated volume is completed. In some embodiments, the transfer of a volume of separately generated polynucleotide:transfer reagent complexes is initiated between approximately 5 minutes and approximately 60 minutes after the transfer of the preceding separately generated volume is completed. In some embodiments, the transfer of a volume of separately generated polynucleotide:transfer reagent complexes is initiated within approximately 5 minutes after the transfer of the preceding separately generated volume is completed. In some embodiments, the transfer of volumes of separately generated polynucleotide:transfer reagent complexes is initiated within approximately 10 minutes after the completion of the transfer of preceding separately generated volumes. In some embodiments, the transfer of volumes of separately generated polynucleotide:transfer reagent complexes is initiated within approximately 15 minutes after the completion of the transfer of preceding separately generated volumes. In some embodiments, the transfer of volumes of separately generated polynucleotide:transfer reagent complexes is initiated within approximately 20 minutes after the completion of the transfer of preceding separately generated volumes. In some embodiments, the transfer of volumes of separately generated polynucleotide:transfer reagent complexes is initiated within approximately 30 minutes after the completion of the transfer of preceding separately generated volumes. In some embodiments, the transfer of volumes of separately generated polynucleotide:transfer reagent complexes is initiated within approximately 45 minutes after the completion of the transfer of preceding separately generated volumes.In some embodiments, the transfer of the volume of the separately generated polynucleotide:transfer reagent complex is initiated within approximately 60 minutes after the completion of the transfer of the preceding separately generated volume. In some embodiments, one or more polynucleotides contain genes necessary for the generation of recombinant AAV particles. In some embodiments, the transfer reagent contains PEI. In some embodiments, the culture contains HEK293 cells (e.g., HEK293 cells adapted for suspension culture).

[0109] In some embodiments, separately generated volumes of polynucleotide:transfer reagent complex are transferred to a cell culture over a period of about 1 to 12 hours. In some embodiments, separately generated volumes of polynucleotide:transfer reagent complex are transferred to a cell culture over a period of about 1 to 8 hours. In some embodiments, separately generated volumes of polynucleotide:transfer reagent complex are transferred to a cell culture over a period of about 1 to 6 hours. In some embodiments, separately generated volumes of polynucleotide:transfer reagent complex are transferred to a cell culture over a period of about 1 to 5 hours. In some embodiments, separately generated volumes of polynucleotide:transfer reagent complex are transferred to a cell culture over a period of about 1 to 4 hours. In some embodiments, separately generated volumes of polynucleotide:transfer reagent complex are transferred to a cell culture over a period of about 1 to 3 hours. In some embodiments, separately generated volumes of polynucleotide:transfer reagent complex are transferred to a cell culture over a period of about 12 hours or less. In some embodiments, separately generated volumes of polynucleotide:transfer reagent complex are transferred to a cell culture over a period of approximately 9 hours or less. In some embodiments, separately generated volumes of polynucleotide:transfer reagent complex are transferred to a cell culture over a period of approximately 8 hours or less. In some embodiments, separately generated volumes of polynucleotide:transfer reagent complex are transferred to a cell culture over a period of approximately 7 hours or less. In some embodiments, separately generated volumes of polynucleotide:transfer reagent complex are transferred to a cell culture over a period of approximately 6 hours or less. In some embodiments, separately generated volumes of polynucleotide:transfer reagent complex are transferred to a cell culture over a period of approximately 5 hours or less. In some embodiments, separately generated volumes of polynucleotide:transfer reagent complex are transferred to a cell culture over a period of approximately 5 hours or less.In some embodiments, separately generated volumes of polynucleotide:transfer reagent complexes are transferred to a cell culture over a period of approximately 3 hours or less. In some embodiments, separately generated volumes of polynucleotide:transfer reagent complexes are transferred to a cell culture over a period of approximately 2 hours or less. In some embodiments, one or more polynucleotides encode genetic information (including genes) necessary for the production of recombinant AAV particles. In some embodiments, one or more polynucleotides include one or more helper genes, rep genes, cap genes, and transgenes (e.g., the target gene or the rAAV genome to be packaged). In some embodiments, the transfer reagent includes PEI. In some embodiments, the culture includes HEK293 cells (e.g., HEK293 cells adapted for suspension culture).

[0110] In some embodiments, the cell culture contains between approximately 2 × 10⁶ and approximately 10⁷ viable cells / ml. In some embodiments, the cell culture contains between approximately 3 × 10⁶ and approximately 8 × 10⁶ viable cells / ml. In some embodiments, the cell culture contains approximately 3 × 10⁶ viable cells / ml. In some embodiments, the cell culture contains approximately 4 × 10⁶ viable cells / ml. In some embodiments, the cell culture contains approximately 5 × 10⁶ viable cells / ml. In some embodiments, the cell culture contains approximately 6 × 10⁶ viable cells / ml. In some embodiments, the cell culture contains approximately 7 × 10⁶ viable cells / ml. In some embodiments, the cell culture contains approximately 8 × 10⁶ viable cells / ml. In some embodiments, the culture contains HEK293 cells (e.g., HEK293 cells adapted for suspension culture).

[0111] In some embodiments, the cell population includes a population of mammalian cells or insect cells. In some embodiments, the cell population includes a population of mammalian cells. In some embodiments, the cell population includes a population of HEK293 cells, HEK-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, and / or PerC6 cells. In some embodiments, the cell population includes a population of HEK293 cells.

[0112] In some embodiments, the cell culture is maintained for about 2 to 10 days after transferring the polynucleotide:transfer reagent complex. In some embodiments, the cell culture is maintained for about 3 to 5 days after transferring the polynucleotide:transfer reagent complex. In some embodiments, the cell culture is maintained for about 5 to 14 days or longer after transferring the polynucleotide:transfer reagent complex. In some embodiments, the cell culture is maintained for about 2 to 7 days after transferring the polynucleotide:transfer reagent complex. In some embodiments, the cell culture is maintained for about 3 to 5 days after transferring the polynucleotide:transfer reagent complex. In some embodiments, the cell culture is maintained for about 2, 3, 4, 5, 6, or 7 days after transferring the polynucleotide:transfer reagent complex. In some embodiments, the cell culture is maintained for at least about 3 days after transferring the polynucleotide:transfer reagent complex. In some embodiments, the cell culture is maintained for about 5 days after transferring the polynucleotide:transfer reagent complex. In some embodiments, the cell culture is maintained for approximately 6 days after transferring the polynucleotide:transfer reagent complex. In some embodiments, the cell culture is maintained under conditions that allow for the generation of rAAV particles for continuous collection. In some embodiments, the culture contains HEK293 cells (e.g., HEK293 cells adapted for suspension culture).

[0113] In some embodiments, the methods disclosed herein increase the production of recombinant virus particles (e.g., rAAV particles) compared to a reference method comprising a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfer reagent complex. In some embodiments, the methods disclosed herein increase the production of recombinant virus by at least about 50%, at least about 75%, or at least about 100%. In some embodiments, the methods disclosed herein increase the production of recombinant virus by at least about 2x, at least about 3x, or at least about 5x. In some embodiments, the methods disclosed herein increase the production of rAAV by at least about 2x. In some embodiments, the increase in production is quantified by comparing the recombinant virus (e.g., rAAV) titer of the produced culture. In some embodiments, the recombinant virus (e.g., rAAV) titer is measured as genome copies (GC) per milliliter of the produced culture. In some embodiments, the recombinant virus is rAAV. In some embodiments, the rAAV particles contain a capsid protein from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV9. In some embodiments, the rAAV particles have a capsid serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles have a capsid protein with high sequence homology to AAV8 or AAV9, for example, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0114] In some embodiments, the methods disclosed herein increase the yield of rAAV particles while maintaining or improving the quality attributes of rAAV particles and compositions containing them. In some embodiments, the quality of rAAV particles and compositions containing them is evaluated by quantifying the concentration of rAAV particles (e.g., GC / ml), the percentage of particles containing copies of the rAAV genome, the proportion of particles without the genome, the infectivity of the rAAV particles, the stability of the rAAV particles, and the concentration of residual host cell proteins or residual host cell nucleic acids (e.g., host cell genomic DNA, plasmids encoding rep and cap genes, plasmids encoding helper functions, plasmids encoding the rAAV genome). In some embodiments, the quality of rAAV particles or compositions containing them produced by the methods disclosed herein is the same as the quality of rAAV particles or compositions produced by a reference method comprising a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfer reagent complex. In some embodiments, the quality of rAAV particles or compositions containing the same produced by the methods disclosed herein is better than the quality of rAAV particles or compositions produced by a reference method which involves a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfer reagent complex.

[0115] In some embodiments, the methods disclosed herein produce rAAV particles between approximately 1 × 10⁻¹⁰ GC / ml and approximately 1 × 10⁻¹³ GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles between approximately 1 × 10⁻¹⁰ GC / ml and approximately 1 × 10⁻¹¹ GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles between approximately 5 × 10⁻¹⁰ GC / ml and approximately 1 × 10⁻¹² GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles between approximately 5 × 10⁻¹⁰ GC / ml and approximately 1 × 10⁻¹³ GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles between approximately 1 × 10⁻¹¹ GC / ml and approximately 1 × 10⁻¹³ GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles between approximately 5 × 10⁻¹⁰ GC / ml and approximately 5 × 10⁻¹² GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles between approximately 1 × 10⁻¹¹ GC / ml and approximately 5 × 10⁻¹² GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles greater than approximately 1 × 10⁻¹¹ GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles greater than approximately 5 × 10⁻¹¹ GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles greater than approximately 1 × 10⁻¹² GC / ml. In some embodiments, the rAAV particles contain a capsid protein from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV9. In some embodiments, the rAAV particles contain a capsid protein from an AAV capsid serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8.In some embodiments, the rAAV particles include capsid proteins with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0116] In some embodiments, the methods disclosed herein produce at least about 5 × 10⁻¹⁰ GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 1 × 10⁻¹¹ GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 5 × 10⁻¹¹ GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 1 × 10⁻¹² GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 5 × 10⁻¹² GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 1 × 10⁻¹³ GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 5 × 10⁻¹³ GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 5 × 10⁻¹³ GC / ml of rAAV particles. In some embodiments, the rAAV particles comprise a capsid protein from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV9. In some embodiments, the rAAV particles contain a capsid protein from an AAV capsid serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles contain a capsid protein with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0117] In the art, numerous cell culture-based systems for the generation of rAAV particles are known, and any of these can be used in carrying out the methods disclosed herein. An rAAV-generating culture for generating rAAV virus particles requires (1) suitable host cells (e.g., human cell lines (e.g., HeLa, A549, or HEK293 cells and their derivatives (HEK293T cells, HEK293F cells)), mammalian cell lines (e.g., Vero), CHO cells or CHO-derived cells); (2) suitable helper virus functions provided by wild-type or mutant adenoviruses (e.g., temperature-sensitive adenoviruses), herpesviruses, baculoviruses, or plasmid constructs that provide helper functions; (3) AAV rep and cap genes and gene products; (4) transgenes adjacent to the AAV ITR sequence (e.g., therapeutic transgenes); and (5) suitable media and media components to support rAAV generation.

[0118] Those skilled in the art are aware of numerous methods by which rAAV can be generated or packaged by introducing AAV rep and cap genes, AAV helper genes (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and rAAV genomes (containing one or more target genes adjacent to reverse terminal repeats (ITRs)) into cells. The expression "adenovirus helper function" means multiple viral helper genes that are expressed intracellularly (as RNA or protein) to enable AAV to grow efficiently within the cell. Those skilled in the art understand that helper viruses, including adenoviruses and herpes simplex viruses (HSV), promote AAV replication, and that certain genes providing essential functions have been identified, and that, for example, helpers can induce changes in the cellular environment that promote such AAV gene expression and replication. In some embodiments of the methods disclosed herein, the AAV rep and cap genes, helper genes, and rAAV genome are introduced into cells by transduction of one or more plasmid vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome.

[0119] Molecular biology techniques for developing plasmids or viral vectors encoding AAV rep and cap genes, helper genes, and / or the rAAV genome are commonly known in the art. In some embodiments, the AAV rep and cap genes are encoded by a single plasmid vector. In some embodiments, AAV helper genes (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes) are encoded by a single plasmid vector. In some embodiments, the E1a or E1b gene is stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transduction with a single viral vector. In some embodiments, the E1a and E1b genes are stably expressed by the host cell, and the E4, E2a, and VA genes are introduced into the cell by transduction with a single plasmid vector. In some embodiments, one or more helper genes are stably expressed by the host cell, and one or more helper genes are introduced into the cell by transduction with a single plasmid vector. In some embodiments, helper genes are stably expressed by host cells. In some embodiments, the AAV rep and cap genes are encoded by a single viral vector. In some embodiments, AAV helper genes (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes) are encoded by a single viral vector. In some embodiments, the E1a or E1b gene is stably expressed by host cells, and the remaining AAV helper genes are introduced into cells by transduction using a single viral vector. In some embodiments, the E1a and E1b genes are stably expressed by host cells, and the E4, E2a, and VA genes are introduced into cells by transduction using a single viral vector. In some embodiments, one or more helper genes are stably expressed by host cells, and one or more helper genes are introduced into cells by transduction using a single viral vector.In some embodiments, the AAV rep and cap genes, the adenovirus helper function required for packaging, and the rAAV genome to be packaged are introduced into cells by transduction using one or more polynucleotides, for example, a vector. In some embodiments, the method disclosed herein involves transduction into cells of a mixture of three polynucleotides (one encoding the cap and rep genes, one encoding the adenovirus helper function required for packaging (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and one encoding the rAAV genome to be packaged). In some embodiments, the AAV cap gene is the AAV8 or AAV9 cap gene. In some embodiments, the AAV cap gene is the AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, or AAV.7m8 cap gene. In some embodiments, the AAV cap gene encodes a capsid protein with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the vector encoding the rAAV genome to be packaged contains the target gene adjacent to the AAV ITR.In some embodiments, AAV ITR is AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2 It is from .5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or another AAV serotype.

[0120] Any combination of vectors can be used to introduce AAV rep and cap genes, AAV helper genes, and the rAAV genome into cells into which rAAV particles are generated or packaged. In some embodiments of the methods disclosed herein, a first plasmid vector encoding the rAAV genome containing a target gene adjacent to an AAV reverse terminal repeat (ITR), a second vector encoding the AAV rep and cap genes, and a third vector encoding the helper gene can be used. In some embodiments, a mixture of the three vectors can be simultaneously translocated into cells.

[0121] In some embodiments, a combination of translocation and infection is used by using a viral vector in conjunction with a plasmid vector.

[0122] In some embodiments, one or more of the rep and cap genes, as well as an AAV helper gene, are constitutively expressed by the cell and do not require transduction or transfection into the cell. In some embodiments, the cell constitutively expresses the rep and / or cap gene. In some embodiments, the cell constitutively expresses one or more AAV helper genes. In some embodiments, the cell constitutively expresses E1a. In some embodiments, the cell contains a stable transgene encoding the rAAV genome.

[0123] In some embodiments, the AAV rep, cap, and helper genes (e.g., Ela gene, E1b gene, E4 gene, E2a gene, or VA gene) can be any AAV serotype. Similarly, the AAV ITR can also be any AAV serotype. For example, in some embodiments, AAV ITR is AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10 , AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B These are from AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or other AAV serotypes (e.g., hybrid serotypes having sequences from two or more serotypes). In some embodiments, the AAV cap gene is from the AAV9 or AAV8 cap gene.In some embodiments, the AAV cap gene is AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3 These are from AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or other AAV serotypes (e.g., hybrid serotypes having sequences from two or more serotypes). In some embodiments, the AAV rep and cap genes for rAAV particle generation are from different serotypes. For example, the rep gene is from AAV2, while the cap gene is from AAV9.

[0124] Any suitable culture medium known in the art can be used to generate recombinant viral particles (e.g., rAAV particles) according to the methods disclosed herein. Such media include, but are not limited to, modified Eagle medium (MEM), Dulbecco's modified Eagle medium (DMEM), and media produced by Hydrone Laboratories and JRH, including Sf-900 II SFM medium as described in U.S. Patent No. 6,723,551 (which is incorporated herein by reference in its entirety). In some embodiments, the medium includes Dynamis® medium, FreeStyle® 293 expression medium, or Expi293® expression medium from Invitrogen / ThermoFisher. In some embodiments, the medium includes Dynamis® medium. In some embodiments, the methods disclosed herein use cell cultures including serum-free medium, animal component-free medium, or chemically defined medium. In some embodiments, the medium is animal component-free medium. In some embodiments, the medium includes serum. In some embodiments, the medium includes fetal bovine serum. In some embodiments, the culture medium is glutamine-free. In some embodiments, the culture medium contains glutamine. In some embodiments, the culture medium is supplemented with one or more of the following: nutrients, salts, buffers, and additives (e.g., antifoaming agents). In some embodiments, the culture medium is supplemented with glutamine. In some embodiments, the culture medium is supplemented with serum. In some embodiments, the culture medium is supplemented with fetal bovine serum. In some embodiments, the culture medium is supplemented with poloxamer, for example, Kolliphor® P 188 Bio. In some embodiments, the culture medium is a basic medium. In some embodiments, the culture medium is a feed medium.

[0125] Recombinant virus (e.g., rAAV) cultures can be routinely grown under a variety of conditions suitable for the specific host cell being used (over a wide temperature range, for varying lengths of time, etc.). As is known in the art, rAAV virus cultures can be adapted to suspension-adapted host cells, such as HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, and TCM cells. These include K-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells, and SF-9 cells, which can be cultured in a variety of ways, including disposable systems such as spinner flasks, agitated tank bioreactors, and wave bag systems. Numerous suspension cultures for generating rAAV particles are known in the art, including, for example, the cultures disclosed in U.S. Patent No. 6,995,006, No. 9,783,826, and U.S. Patent Application Publication No. 20120122155 (each of which is incorporated herein by reference in whole). In some embodiments, the recombinant virus is recombinant AAV.

[0126] Any cell or cell line known in the Art to produce recombinant viral particles (e.g., rAAV particles) can be used in any one of the methods disclosed herein. In some embodiments, the methods disclosed herein for producing or increasing the production of recombinant viral particles (e.g., rAAV particles) use HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, LLC-MK cells, MDCK cells, RAF cells, RK cells, TCMK-1 cells, PK15 cells, BHK cells, BHK-21 cells, NS-1 cells, BHK cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells, or SF-9 cells. In some embodiments, the methods disclosed herein use mammalian cells. In some embodiments, the methods disclosed herein use insect cells, such as SF-9 cells. In some embodiments, the methods disclosed herein use cells adapted for growth in suspension culture. In some embodiments, the methods disclosed herein use HEK293 cells adapted for growth in suspension culture. In some embodiments, recombinant virus particles are recombinant AAV particles.

[0127] In some embodiments, the cell cultures disclosed herein are suspension cultures. In some embodiments, the large-scale suspension cell cultures disclosed herein contain HEK293 cells adapted for growth in suspension culture. In some embodiments, the cell cultures disclosed herein contain serum-free medium, animal component-free medium, or chemically defined medium. In some embodiments, the cell cultures disclosed herein contain serum-free medium. In some embodiments, suspension-adapted cells are cultured in a shaking flask, spinner flask, cell bag, or bioreactor.

[0128] In some embodiments, the cell cultures disclosed herein include serum-free media, animal component-free media, or chemically defined media. In some embodiments, the cell cultures disclosed herein include serum-free media.

[0129] In some embodiments, the cell cultures disclosed herein contain an anti-aggregating agent. In some embodiments, the cell cultures disclosed herein contain dextran sulfate. In some embodiments, the cell cultures disclosed herein contain dextran sulfate in a concentration between about 0.1 mg / L and about 10 mg / L. A method for transposing host cells in a culture medium containing dextran sulfate is disclosed in U.S. Provisional Application No. 63 / 139,992, titled "Improved production of recombinant polypeptides and viruses," filed on 21 January 2021 (the entire application of which is incorporated herein by reference).

[0130] In some embodiments, the large-scale suspension cell cultures disclosed herein include high-density cell cultures. In some embodiments, the culture has a total cell density between about 1 × 10⁻⁶ cells / ml and about 30 × 10⁻⁶ cells / ml. In some embodiments, more than about 50% of the cells are viable cells. In some embodiments, the cells are HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, or SF-9 cells. In further embodiments, the cells are HEK293 cells.

[0131] The methods disclosed herein can be used to generate rAAV particles containing capsid proteins from any AAV capsid serotype. In some embodiments, the rAAV particles are AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, It contains capsid proteins from AAV capsid serotypes selected from AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, rAAV particles are AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5 This includes capsid proteins that are derivatives, modifiers, or pseudotypes of AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 capsid proteins.

[0132] In some embodiments, the rAAV particles contain a capsid protein from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV9.

[0133] In some embodiments, the rAAV particles contain a capsid protein from an AAV capsid serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles contain a capsid protein with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0134] In some embodiments, the rAAV particles contain a capsid protein that is a derivative, modifier, or pseudotype of the AAV8 capsid protein or the AAV9 capsid protein. In some embodiments, the rAAV particles contain a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV8 capsid protein, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical to the AAV8 capsid protein.

[0135] In some embodiments, the rAAV particles contain a capsid protein that is a derivative, modifier, or pseudotype of the AAV9 capsid protein. In some embodiments, the rAAV particles contain a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV9 capsid protein, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical to the AAV9 capsid protein.

[0136] In some embodiments, the rAAV particles contain capsid proteins having at least 80% identity to the VP1, VP2, and / or VP3 sequences of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, or AAV.7m8 capsid proteins, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identity. In some embodiments, rAAV particles contain AAV capsid proteins with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37, with at least 80% identity to the VP1, VP2, and / or VP3 sequences, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, i.e., capsid proteins with up to 100% identity.

[0137] In additional embodiments, the rAAV particles include a mosaic capsid. In additional embodiments, the rAAV particles include pseudotype rAAV particles. In additional embodiments, the rAAV particles include a capsid containing a capsid protein chimera of two or more AAV capsid serotypes.

[0138] rAAV particles The method provided is suitable for use in the generation of any isolated recombinant AAV particles. Thus, rAAV can be any serotype, modifier, or derivative known in the art, or any combination thereof (e.g., a population of rAAV particles containing two or more serotypes (e.g., two or more of rAAV2, rAAV8, and rAAV9 particles)). In some embodiments, rAAV particles are AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, A AV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or other rAAV particles, or a combination of two or more of these.

[0139] In some embodiments, rAAV particles are AAV1, AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV It possesses a capsid protein from an AAV serotype selected from 2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or their derivatives, modifications, or pseudotypes.In some embodiments, rAAV particles are, for example, AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, rAAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, A The capsid protein contains a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of an AAV capsid serotype selected from AV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical.

[0140] In some embodiments, rAAV particles are AAV1, AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2t This includes capsid proteins from AAV capsid serotypes selected from YF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or their derivatives, modifiers, or pseudotypes.In some embodiments, rAAV particles are, for example, AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, A The capsid protein contains a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of an AAV capsid serotype selected from AV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical.

[0141] In some embodiments, the rAAV particles comprise a capsid of Anc80 or Anc80L65, as described in Zinn et al., 2015, Cell Rep. 12(6):1056-1068 (which is incorporated herein by reference in its entirety). In certain embodiments, the rAAV particles comprise a capsid having one of the amino acid inserts: LGETTRP or LALGETTRP, as described in U.S. Patents 9,193,956, 9,458,517, and 9,587,282, and U.S. Patent Application Publication 2016 / 0376323 (each of which is incorporated herein by reference in its entirety). In some embodiments, the rAAV particles include the capsid of AAV.7m8, as described in U.S. Patent Nos. 9,193,956, 9,458,517, and 9,587,282, and U.S. Patent Application Publication No. 2016 / 0376323 (each of which is incorporated herein by reference in whole). In some embodiments, the rAAV particles include any AAV capsid disclosed in U.S. Patent No. 9,585,971, e.g., AAVPHP.B. In some embodiments, the rAAV particles include any AAV capsid disclosed in U.S. Patent No. 9,840,719 and WO2015 / 013313 (each of which is incorporated herein by reference in whole), e.g., AAV.Rh74 and RHM4-1. In some embodiments, the rAAV particles include any AAV capsid disclosed in WO2014 / 172669 (which is incorporated herein by reference in its entirety), e.g., AAV rh.74. In some embodiments, the rAAV particles include the AAV2 / 5 capsid as described in Georgiadis et al., 2016, Gene Therapy 23:857-862 and Georgiadis et al., 2018, Gene Therapy 25:450 (each of which is incorporated herein by reference in its entirety). In some embodiments, the rAAV particles include any AAV capsid disclosed in WO2017 / 070491 (which is incorporated herein by reference in its entirety), e.g., AAV2tYF.In some embodiments, the rAAV particles include a capsid of AAVLK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9):418 (each of which is incorporated herein by reference in whole). In some embodiments, the rAAV particles include any AAV capsid disclosed in U.S. Patent Nos. 8,628,966, U.S. 8,927,514, U.S. 9,923,120, and WO2016 / 049230, e.g., HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16 (each of which is incorporated herein by reference in whole).

[0142] In some embodiments, rAAV particles are used in the following patents and patent applications (each of which is incorporated herein by reference in whole): U.S. Patent Nos. 7,282,199, 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, U.S. Patent Nos. 9,284,357, 9,409,953, 9,169,299, 9,193, This includes AAV capsids disclosed in any of the following: Patent Nos. 956, 9458517, and 9,587,282; U.S. Patent Application Publications 2015 / 0374803, 2015 / 0126588, 2017 / 0067908, 2013 / 0224836, 2016 / 0215024, 2017 / 0051257; and International Patent Application Nos. PCT / US2015 / 034799 and PCT / EP2015 / 053335. In some embodiments, the rAAV particles have a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV capsid disclosed in any of the following patents and patent applications (each of which is incorporated herein by reference in whole): for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical: U.S. Patents 7,282,199, 7,906,111, 8,524,446, 8,9 U.S. Patent Applications Nos. 99,678, 8,628,966, 8,927,514, 8,734,809, U.S. Patent Nos. 9,284,357, 9,409,953, 9,169,299, 9,193,956, 9,458,517, and 9,587,282, U.S. Patent Application Publication No. U.S.2015 Patent applications No. / 0374803, No. 2015 / 0126588, No. 2017 / 0067908, No. 2013 / 0224836, No. 2016 / 0215024, No. 2017 / 0051257, and international patent applications PCT / US2015 / 034799 and PCT / EP2015 / 053335.

[0143] In some embodiments, rAAV particles are used in International Patent Application Publications WO2003 / 052051 (see, e.g., SEQ ID NO: 2), WO2005 / 033321 (see, e.g., SEQ ID NOs: 123 and 88), WO03 / 042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97), WO2006 / 068888 (see, e.g., SEQ ID NOs: 1 and 3-6), WO2006 / 110689 (see, e.g., SEQ ID NOs: 5-38), WO2009 / 1 The capsid protein is disclosed in Patent No. 04964 (see, for example, SEQ ID NOs. 1-5, 7, 9, 20, 22, 24, and 31), WO2010 / 127097 (see, for example, SEQ ID NOs. 5-38), and WO2015 / 191508 (see, for example, SEQ ID NOs. 80-294), and U.S. Patent Application Publication No. 20150023924 (see, for example, SEQ ID NOs. 1, 5-10) (each of which is incorporated herein by reference in its entirety). In some embodiments, the rAAV particles have a capsid protein that is at least 80% identical to the VP1, VP2 and / or VP3 sequences of the AAV capsid disclosed below, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, i.e., up to 100% identical: International Patent Application Publication WO2003 / 052051 (see, e.g., SEQ ID NO: 2), WO2005 / 033321 (see, e.g., SEQ ID NOs: 123 and 88), WO03 / 04239 See, for example, Sequence IDs 2, 81, 85, and 97; WO2006 / 068888 (see, for example, Sequence IDs 1 and 3-6); WO2006 / 110689 (see, for example, Sequence IDs 5-38); WO2009 / 104964 (see, for example, Sequence IDs 1-5, 7, 9, 20, 22, 24, and 31); WO2010 / 127097 (see, for example, Sequence IDs 5-38); and WO2015 / 191508 (see, for example, Sequence IDs 80-294); and U.S. Patent Application Publication No. 20150023924 (see, for example, Sequence IDs 1, 5-10).

[0144] Nucleic acid sequences of AAV-based viral vectors, as well as methods for producing recombinant AAV and AAV capsids, are taught, for example, in: U.S. Patents Nos. 7,282,199, 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, U.S. Patents Nos. 9,284,357, 9,409,953, 9,169,299, 9,193,956, 9458517, and 9,587,282, U.S. Patent Application Publications 2015 / 0374803 and 2015 / 0126588. Applications No. 2017 / 0067908, No. 2013 / 0224836, No. 2016 / 0215024, No. 2017 / 0051257, International Patent Application No. PCT / US2015 / 034799, No. PCT / EP2015 / 053335, No. WO2003 / 052051, No. WO2005 Patent applications Nos. / 033321, WO03 / 042397, WO2006 / 068888, WO2006 / 110689, WO2009 / 104964, WO2010 / 127097, and WO2015 / 191508, as well as U.S. Patent Application Publication No. 20150023924.

[0145] The provided method is suitable for use in the generation of recombinant AAV encoding a transgene. In certain embodiments, the transgene is selected from Tables 2A-2C. In some embodiments, the rAAV genome comprises a vector containing the following components: (1) AAV reverse terminal repeats adjacent to the expression cassette, (2) regulatory elements, e.g., a) promoter / enhancer, b) polyA signal, and c) introns, optionally, and (3) a nucleic acid sequence encoding the transgene. In other embodiments for expressing intact or substantially intact monoclonal antibodies (mAbs), the rAAV genome comprises a vector containing the following components: (1) AAV reverse terminal repeats adjacent to the expression cassette, (2) regulatory elements, e.g., a) promoter / enhancer, b) polyA signal, and c) introns, optionally, and (3) a nucleic acid sequence encoding the light chain Fab and heavy chain Fab of the antibody, or at least the heavy chain or light chain Fab, and optionally the heavy chain Fc region. In yet another embodiment for expressing intact or substantially intact mAbs, the rAAV genome comprises a vector comprising: (1) AAV reverse terminal repeats adjacent to the expression cassette; (2) regulatory elements, e.g., a) promoter / enhancer, b) polyA signaling, and c) introns of choice;(3) Nucleic acid sequences encoding heavy chain Fab of the following: anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651), anti-ALK1 (e.g., askrinbakumab), anti-C5 (e.g., tesidolumab and eculizumab), anti-CD105 (e.g., carotuximab), anti-CC1Q (e.g., ANX-007), anti-TNFα (e.g., adalimumab, infliximab, and golim) Anti-IL-5 (e.g., mepolizumab), anti-IL-12 / IL-23 (e.g., ustekinumab), anti-IL-19 (e.g., inebilizumab), anti-IL-19 (e.g., inebilizumab), anti-IL-19 (e.g., elezanumab), anti-IL-19 (e.g., lezanumab), anti-IL-19 (e.g., inebilizumab), anti-IL-19 (e.g., inebilizumab), anti-IL-19 (e.g., elezan mAb (e.g., etrolizumab), anti-SOST mAb (e.g., romosozumab), anti-pKal mAb (e.g., lanadelmab), anti-ITGA4 (e.g., natalizumab), anti-ITGA4B7 (e.g., vedolizumab), anti-BLyS (e.g., belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab), anti-RANKL (e.g., densomab), anti-PCSK9 (e.g., alirocumab and evolocumab), anti-ANGPTL3 (e.g., evinacumab*), anti-OxPL (e.g., E06), anti-fD (e.g., lamparizumab), or anti-MMP9 (e.g., andecaliximab); optionally, an Fc polypeptide of the same isotype as the native form of the therapeutic antibody, e.g., IgG isotype amino acid sequence IgG1, IgG2, or IgG4, or modified Fc thereof;Furthermore, nucleic acid sequences encoding the light chains of the following: anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651), anti-ALK1 (e.g., askrinbakumab), anti-C5 (e.g., tesidolumab and eculizumab), anti-CD105 or anti-ENG (e.g., carotuximab), anti-CC1Q (e.g., ANX-007), anti-TNFα (e.g., adalimumab, infliximab, and goli Mumab), anti-RGMa (e.g., elezanumab), anti-TTR (e.g., NI-301 and PRX-004), anti-CTGF (e.g., pamlevlumab), anti-IL6R (e.g., satralizumab and sarilumab), anti-IL4R (e.g., dupilumab), anti-IL17A (e.g., ixekizumab and secukinumab), anti-IL-5 (e.g., mepolizumab), anti-IL12 / IL23 (e.g., ustekinumab), anti-CD19 (e.g., inebilizumab), anti-ITGF7 mAbs (e.g., etrolizumab), anti-SOST mAbs (e.g., romosozumab), anti-pKal mAbs (e.g., lanadelmab), anti-ITGA4 (e.g., natalizumab), anti-ITGA4B7 (e.g., vedolizumab), anti-BLyS (e.g., belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab), anti-RANKL (e.g., densomab), anti-PCSK9 (e.g., alirocumab and evolocumab), anti-ANGPTL3 (e.g., evinacumab), anti-OxPL (e.g., E06), anti-fD (e.g., lampalizumab), or anti-MMP9 (e.g., andecaliximab). At this time, the heavy chain (Fab and optionally the Fc region) and light chain are separated by self-cleaving furin(F) / F2A or furin(F) / F2A or a flexible linker, ensuring that the heavy chain and light chain polypeptides are expressed in equal amounts.

[0146] [Table 2A] TIFF2023554389000003.tif239165TIFF2023554389000004.tif224165TIFF2023554389000005.tif220165TIFF2023554389000006.tif219165TIFF2023554389000007.tif39165

[0147]

Table 2B

[0148]

Table 2C

[0149] In some embodiments, rAAV viral vectors encoding anti-VEGF Fab are provided herein. In specific embodiments, rAAV8-based viral vectors encoding anti-VEGF Fab are provided herein. In more specific embodiments, rAAV8-based viral vectors encoding ranibizumab are provided herein. In some embodiments, rAAV viral vectors encoding idulonidase (IDUA) are provided herein. In specific embodiments, rAAV9-based viral vectors encoding IDUA are provided herein. In some embodiments, rAAV viral vectors encoding iduronate 2-sulfatase (IDS) are provided herein. In specific embodiments, rAAV9-based viral vectors encoding IDS are provided herein. In some embodiments, rAAV viral vectors encoding low-density lipoprotein receptor (LDLR) are provided herein. In specific embodiments, rAAV8-based viral vectors encoding LDLR are provided herein. In some embodiments, rAAV viral vectors encoding tripeptidyl peptidase 1 (TPP1) protein are provided herein. In specific embodiments, rAAV9-based viral vectors encoding TPP1 are provided herein. In some embodiments, rAAV viral vectors encoding a non-membrane-bound splice variant of VEGF receptor 1 (sFlt-1) are provided herein.In some embodiments, the following are used herein: gamma-sarcoglycan, Rab escort protein 1 (REP1 / CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide gate channel alpha-3 (CNGA3), cyclic nucleotide gate channel beta-3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosome-associated membrane protein 2 isoform B (LAMP2B), factor VIII, factor IX, retinitis pigmentosa GTPase regulator (RPGR), retinoskin (RS1). , sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamate decarboxylase (GAD), glial cell line-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, microdystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyltransferase 1A1 (U GT1A1), arylsulfatase B (ARSB), N-acetyl-alpha-glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha-1-antitrypsin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN1), survival motor neuron (SMN2), neuruturin (NRTN), neutrinopropyltrione rAAV viral vectors are provided that encode -trotrophin-3 (NT-3 / NTF3), porphobilinogen deaminase (PBGD), nerve growth factor (NGF), mitochondrial-coded NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dyspherin, MER proto-oncogene tyrosine kinase (MERTK), cystic fibrosis membrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgG1)Fc fusion.

[0150] In additional embodiments, the rAAV particles comprise a pseudotype AAV capsid. In some embodiments, the pseudotype AAV capsid is an rAAV2 / 8 or rAAV2 / 9 pseudotype AAV capsid. Methods for generating and using pseudotype rAAV particles are known in the art (see, for example, Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081 (2001)).

[0151] In additional embodiments, the rAAV particles contain a capsid comprising a capsid protein that is a chimeric of two or more AAV capsid serotypes. In some embodiments, the capsid protein is AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, A It is a chimera of two or more AAV capsid proteins from AAV serotypes selected from AV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[0152] In certain embodiments, single-stranded AAVs (ssAAVs) may be used. In certain embodiments, self-complementary vectors, such as scAAVs, may be used (see, for example, Wu, 2007, Human Gene Therapy, 18(2):171-82; McCarty et al, 2001, Gene Therapy, Vol. 8, Number 16:1248-1254; and U.S. Patents 6,596,535, 7,125,717, and 7,456,683 (each of which is incorporated herein by reference in whole)).

[0153] In some embodiments, the rAAV particles contain a capsid protein from an AAV capsid serotype selected from AAV8 or AAV9. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV9.

[0154] In some embodiments, the rAAV particles contain a capsid protein that is a derivative, modifier, or pseudotype of the AAV8 capsid protein or the AAV9 capsid protein. In some embodiments, the rAAV particles contain a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV8 capsid protein, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical to the AAV8 capsid protein.

[0155] In some embodiments, the rAAV particles contain a capsid protein that is a derivative, modifier, or pseudotype of the AAV9 capsid protein. In some embodiments, the rAAV particles contain a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV9 capsid protein, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical to the AAV9 capsid protein.

[0156] In additional embodiments, rAAV particles include mosaic capsids. Mosaic AAV particles consist of a mixture of viral capsid proteins from different serotypes of AAV. In some embodiments, rAAV particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AA It contains a mosaic capsid containing a capsid protein of a serotype selected from V2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles include a mosaic capsid containing a capsid protein of a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.8, AAVrh.10, AAVrh.37, AAVrh.20, and AAVrh.74.

[0157] In additional embodiments, the rAAV particles include pseudotyped rAAV particles. In some embodiments, the pseudotyped rAAV particles include (a) a nucleic acid vector containing AAV ITR, and (b) AAVx (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.P It contains a capsid composed of capsid proteins derived from HP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16). In additional embodiments, the rAAV particles include pseudotyped rAAV particles composed of capsid proteins of AAV serotypes selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.8, AAVrh.10, AAVhu.37, AAVrh.20, and AAVrh.74. In additional embodiments, the rAAV particles include pseudotyped rAAV particles containing the AAV8 capsid protein. In additional embodiments, the rAAV particles include pseudotyped rAAV particles composed of the AAV9 capsid protein. In some embodiments, the pseudotyped rAAV8 or rAAV9 particles are rAAV2 / 8 or rAAV2 / 9 pseudotyped particles.Methods for generating and using pseudotype rAAV particles are known in the art (see, for example, Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081 (2001)).

[0158] In additional embodiments, the rAAV particles contain a capsid protein that is a chimeric of two or more AAV capsid serotypes. In some embodiments, the rAAV particles contain the AAV8 capsid protein and AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV 2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16, comprising an AAV capsid protein chimera with one or more AAV capsid proteins from AAV serotypes selected from AAV serotypes. In some embodiments, the rAAV particles contain an AAV capsid protein that is a chimera of the AAV8 capsid protein and one or more AAV capsid proteins from AAV serotypes selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV9, AAV10, rAAVrh10, AAVrh.8, AAVrh.10, AAVrh.37, AAVrh.20, and AAVrh.74.In some embodiments, rAAV particles contain the AAV9 capsid protein and AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, and AAV2. 5. Contains an AAV capsid protein that is a chimera with a capsid protein of one or more AAV capsid serotypes selected from AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles contain an AAV capsid protein that is a chimera of the AAV9 capsid protein and the capsid protein of one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAVrh.37, AAVrh.20, and AAVrh.74.

[0159] Method for isolating rAAV particles In some embodiments, the Disclosure provides a method for producing a composition comprising isolated recombinant adeno-associated virus (rAAV) particles, the method comprising isolating rAAV particles from a feed containing impurities (e.g., an rAAV-producing culture). In some embodiments, the method for producing a formulation comprising isolated recombinant adeno-associated virus (rAAV) particles disclosed herein comprises (a) isolating rAAV particles from a feed containing impurities (e.g., an rAAV-producing culture), and (b) formulation the isolated rAAV particles to produce a formulation.

[0160] In some embodiments, the Disclosure further provides a method for producing a formulation comprising isolated recombinant adeno-associated virus (rAAV) particles in a pharmaceutical unit dose, the method comprising isolating rAAV particles from a feed containing impurities (e.g., an rAAV-producing culture) and formulating the isolated rAAV particles.

[0161] The isolated rAAV particles can be isolated using methods known in the art. In some embodiments, the method for isolating rAAV particles includes downstream processing, e.g., collection of cell cultures, clarification of the collected cell cultures (e.g., by centrifugation or deep filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, sterile filtration, or any combination(s) of these. In some embodiments, the downstream processing includes at least two, at least three, at least four, at least five, or at least six of the following: collection of cell cultures, clarification of the collected cell cultures (e.g., by centrifugation or deep filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, and sterile filtration. In some embodiments, the downstream process includes collecting the cell culture, clarifying the collected cell culture (e.g., by deep filtration), sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, the downstream process includes clarifying the collected cell culture, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, the downstream process includes clarifying the collected cell culture by deep filtration, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, the clarification of the collected cell culture includes sterile filtration. In some embodiments, the downstream process does not include centrifugation. In some embodiments, the rAAV particles contain the AAV8 serotype capsid protein. In some embodiments, the rAAV particles contain the AAV9 serotype capsid protein.

[0162] In some embodiments, a method for isolating rAAV particles according to the method disclosed herein includes collecting a cell culture, clarifying the collected cell culture (e.g., by deep filtration), a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolithic anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration. In some embodiments, a method for isolating rAAV particles produced according to the method disclosed herein includes clarification of the collected cell culture, a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolithic anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration.In some embodiments, a method for isolating rAAV particles produced according to the method disclosed herein includes clarification of the collected cell culture by deep filtration, a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolithic anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration. In some embodiments, a method for isolating rAAV particles disclosed herein includes clarification of the collected cell culture by deep filtration, a first sterile filtration, affinity chromatography, anion exchange chromatography (e.g., monolithic anion exchange chromatography or AEX chromatography using a quaternary amine ligand), tangential flow filtration, and a second sterile filtration. In some embodiments, the method does not include centrifugation. In some embodiments, clarification of the collected cell culture includes sterile filtration. In some embodiments, the rAAV particles contain the capsid protein of the AAV8 serotype. In some embodiments, the rAAV particles contain the capsid protein of the AAV9 serotype.

[0163] In this field, numerous cell culture-based systems are known for translocation, stable cell line generation, and the production of rAAV particles, including infectious hybrid virus generation systems (including adenovirus-AAV hybrids, herpesvirus-AAV hybrids, and baculovirus-AAV hybrids). Any rAAV-producing culture for generating rAAV virus particles requires (1) a suitable host cell (e.g., a human cell line (e.g., HeLa, A549, or HEK293 cells and their derivatives (HEK293T cells, HEK293F cells)), a mammalian cell line (e.g., Vero), or, in the case of a baculovirus-producing system, an insect cell line (e.g., SF-9)); (2) a suitable helper virus function provided by a wild-type or mutant adenovirus (e.g., a temperature-sensitive adenovirus), a herpesvirus, a baculovirus, or a plasmid construct providing helper function; (3) AAV rep and cap genes and gene products; (4) a transgene adjacent to the AAV ITR sequence (e.g., a therapeutic transgene); and (5) a suitable medium and medium components to support rAAV production. Suitable media known in the art can be used for the production of rAAV vectors. Such media include, but are not limited to, modified Eagle medium (MEM), Dulbecco's modified Eagle medium (DMEM), and media produced by Hyclone Laboratories and JRH, including Sf-900 II SFM medium as described in U.S. Patent No. 6,723,551 (which is incorporated herein by reference in its entirety).

[0164] rAAV-producing cultures can be routinely grown under various conditions suitable for the specific host cells being used (over a wide temperature range, over varying lengths of time, etc.). As is known in the art, rAAV-producing cultures include adhesion-dependent cultures that can be cultured in suitable adhesion-dependent containers (e.g., roller bottles, hollow fiber filters, microcarriers, and packed or fluidized bed bioreactors). Furthermore, rAAV vector-producing cultures can be grown with suspension-adaptive host cells, such as HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLC The cells may also include PK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells, or SF-9 cells, which can be cultured in a variety of ways, including, for example, spinner flasks, agitated tank bioreactors, and disposable systems such as Wave bag systems. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293 cells adapted for growth in suspension culture. Numerous suspension cultures for generating rAAV particles are known in the Art, including, for example, the cultures disclosed in U.S. Patent No. 6,995,006, No. 9,783,826, and U.S. Patent Application Publication No. 20120122155 (each of which is incorporated herein by reference in whole).

[0165] In some embodiments, the rAAV-producing culture comprises a high-density cell culture. In some embodiments, the culture has a total cell density between approximately 1 × 10⁻⁶ cells / ml and approximately 30 × 10⁻⁶ cells / ml. In some embodiments, more than approximately 50% of the cells are viable cells. In some embodiments, the cells are HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, or SF-9 cells. In further embodiments, the cells are HEK293 cells. In further embodiments, the cells are HEK293 cells adapted for growth in suspension culture.

[0166] In additional embodiments of the provided method, the rAAV-producing culture includes a suspension culture containing rAAV particles. Numerous suspension cultures for producing rAAV particles are known in the Art, including, for example, the cultures disclosed in U.S. Patent No. 6,995,006, No. 9,783,826, and U.S. Patent Application Publication No. 20120122155 (each of which is incorporated herein by reference in whole). In some embodiments, the suspension culture includes a culture of mammalian cells or insect cells. In some embodiments, the suspension culture includes cultures of HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells, or SF-9 cells. In some embodiments, the suspension culture includes a culture of HEK293 cells.

[0167] In some embodiments, a method for generating rAAV particles comprises preparing a cell culture containing cells capable of generating rAAV, adding a histone deacetylase (HDAC) inhibitor to the cell culture to a final concentration between about 0.1 mM and about 20 mM, and maintaining the cell culture under conditions that enable the generation of rAAV particles. In some embodiments, the HDAC inhibitor comprises a short-chain fatty acid or a salt thereof. In some embodiments, the HDAC inhibitor comprises butyric acid (e.g., sodium butyrate), valproic acid (e.g., sodium valproate), propionic acid (e.g., sodium propionate), or a combination thereof.

[0168] In some embodiments, rAAV particles are generated as disclosed in WO2020 / 033842 (which is incorporated herein by reference in its entirety).

[0169] Recombinant AAV particles can be collected from rAAV-producing cultures by collecting the product culture containing host cells, or by collecting the consumed medium from the product culture, provided that the cells are cultured under conditions known in the art to induce the release of rAAV particles from intact host cells into the culture medium. Recombinant AAV particles can also be collected from rAAV-producing cultures by lysing the host cells of the product culture. Suitable methods for lysing cells are also known in the art, and include, for example, multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals (e.g., surfactants and / or proteases).

[0170] At collection, rAAV-producing cultures may contain one or more of the following: (1) host cell proteins; (2) host cell DNA; (3) plasmid DNA; (4) helper viruses; (5) helper virus proteins; (6) helper virus DNA; and (7) culture medium components (e.g., serum proteins, amino acids, transferrin, and other low molecular weight proteins). rAAV-producing cultures may also contain product-related impurities, such as inactive vector forms, empty viral capsids, aggregated viral particles or capsids, misfolded viral capsids, and degraded viral particles.

[0171] In some embodiments, the rAAV-producing culture collection is clarified to remove host cell debris. In some embodiments, the producing culture collection is clarified by filtration through a series of deep filters. Clarification can also be achieved by various other standard techniques known in the art, for example, by centrifugation or by filtration through any cellulose acetate filter with a pore size of 0.2 mm or larger known in the art. In some embodiments, clarification of the collected cell culture includes sterile filtration. In some embodiments, the producing culture collection is clarified by centrifugation. In some embodiments, clarification of the producing culture collection does not include centrifugation.

[0172] In some embodiments, the collected cell culture is clarified using filtration. In some embodiments, the clarification of the collected cell culture includes deep filtration. In some embodiments, the clarification of the collected cell culture further includes deep filtration and sterile filtration. In some embodiments, the collected cell culture is clarified using a filter train containing one or more different filtration media. In some embodiments, the filter train contains one deep filtration medium. In some embodiments, the filter train contains one or more deep filtration media. In some embodiments, the filter train contains two deep filtration media. In some embodiments, the filter train contains one sterile filtration medium. In some embodiments, the filter train contains two deep filtration media and one sterile filtration medium. In some embodiments, the deep filtration medium is a porous deep filter. In some embodiments, the filter train contains Clarisolve® 20MS, Millistak+® C0HC, and sterile-grade filtration media. In some embodiments, the filter train includes Clarisolve® 20MS, Millistak+® C0HC, and Sartopore® 2 XLG 0.2 μm. In some embodiments, the collected cell culture is pretreated before contact with the deep filter. In some embodiments, the pretreatment includes adding salt to the collected cell culture. In some embodiments, the pretreatment includes adding a chemical flocculant to the collected cell culture. In some embodiments, the collected cell culture is not pretreated before contact with the deep filter.

[0173] In some embodiments, the collected culture is clarified by filtration, as disclosed in WO2019 / 212921 (which is incorporated herein by reference in its entirety).

[0174] In some embodiments, rAAV-producing culture collections are treated with a nuclease (e.g., Bensonase®) or endonuclease (e.g., endonuclease from Serratia marcescens) to digest the high molecular weight DNA present in the producing culture. Nuclease or endonuclease digestion can be routinely carried out under standard conditions known in the art. For example, nuclease digestion is carried out for 30 minutes to several hours with a final concentration of 1 to 2.5 units / mL of Bensonase® at temperatures ranging from ambient temperature to 37°C.

[0175] Aseptic filtration encompasses filtration using a sterile-grade filter medium. In some embodiments, the sterile-grade filter medium is a 0.2 or 0.22 μm pore filter. In some embodiments, the sterile-grade filter medium contains polyethersulfone (PES). In some embodiments, the sterile-grade filter medium contains polyvinylidene fluoride (PVDF). In some embodiments, the sterile-grade filter medium has a hydrophilic heterogeneous double-layer design. In some embodiments, the sterile-grade filter medium has a hydrophilic heterogeneous double-layer design of a 0.8 μm pre-filter and a 0.2 μm final filter membrane. In some embodiments, the sterile-grade filter medium has a hydrophilic heterogeneous double-layer design of a 1.2 μm pre-filter and a 0.2 μm final filter membrane. In some embodiments, the sterile-grade filter medium is a 0.2 or 0.22 μm pore filter. In further embodiments, the sterile-grade filter medium is a 0.2 μm pore filter. In some embodiments, the sterile-grade filter medium is a combination of nominal pore sizes of Sartopore® 2 XLG 0.2 μm, Durapore® PVDF membrane 0.45 μm, or Sartoguard® PES 1.2 μm + 0.2 μm. In some embodiments, the sterile-grade filter medium is Sartopore® 2 XLG 0.2 μm.

[0176] In some embodiments, the clarified feed is concentrated via tangential flow filtration ("TFF") before being applied to a chromatographic medium, such as an affinity chromatography medium. Large-scale concentrations of viruses using TFF ultrafiltration are described in Paul et al., Human Gene Therapy 4:609-615 (1993). The TFF concentration of the clarified feed allows for chromatographic application of technically controllable amounts of the clarified feed and enables more rational column sizing without requiring long recirculation times. In some embodiments, the clarified feed is concentrated between at least 2x and at least 10x. In some embodiments, the clarified feed is concentrated between at least 10x and at least 20x. In some embodiments, the clarified feed is concentrated between at least 20x and at least 50x. In some embodiments, the clarified feed is concentrated to about 20x. Those skilled in the art will also recognize that TFF may be used to remove small molecule impurities (e.g., cell culture impurities including culture medium components, serum albumin, or other serum proteins) from a feed clarified via diafiltration. In some embodiments, the clarified feed is subjected to diafiltration to remove small molecule impurities. In some embodiments, the diafiltration includes using a diafiltration volume of buffer between about 3 and about 10. In some embodiments, the diafiltration includes using a diafiltration volume of buffer between about 5. Those skilled in the art will also recognize that TFF may be used at any step of the purification process where it is desirable to exchange the buffer before carrying out the next step in the purification process. In some embodiments, the method for isolating rAAV from a clarified feed disclosed herein includes the use of TFF for buffer exchange.

[0177] Affinity chromatography can be used to isolate rAAV particles from a composition. In some embodiments, affinity chromatography is used to isolate rAAV particles from a clarified feed. In some embodiments, affinity chromatography is used to isolate rAAV particles from a feed that has been clarified by tangential flow filtration. Suitable affinity chromatography media are known in the art and are not limited to AVB Sepharose®, POROS® CaptureSelect® AAVX affinity resin, POROS® CaptureSelect® AAV9 affinity resin, and POROS® CaptureSelect® AAV8 affinity resin. In some embodiments, the affinity chromatography medium is POROS® CaptureSelect® AAV9 affinity resin. In some embodiments, the affinity chromatography medium is POROS® CaptureSelect® AAV8 affinity resin. In some embodiments, the affinity chromatography medium is POROS® CaptureSelect® AAVX affinity resin.

[0178] Anion exchange chromatography can be used to isolate rAAV particles from a composition. In some embodiments, anion exchange chromatography is used after affinity chromatography as a final concentration and polishing step. Suitable anion exchange chromatography media are known in the art and are not limited to, but include UNOsphere® Q (Biorad, Hercules, Calif.) and N-charged amino or imino resins, e.g., POROS® 50 PI, or any DEAE, TMAE, tertiary or quaternary amine, or PEI-based resins known in the art (U.S. Patent No. 6,989,264; Brument et al., Mol. Therapy 6(5):678-686 (2002); Gao et al., Hum. Gene Therapy 11:2079-2091 (2000)). In some embodiments, the anion exchange chromatography media contains a quaternary amine. In some embodiments, the anion exchange medium is a monolithic anion exchange chromatography resin. In some embodiments, the monolithic anion exchange chromatography medium comprises a glycidyl methacrylate-ethylenedimethacrylate polymer or a styrene-divinylbenzene polymer. In some embodiments, the monolithic anion exchange chromatography medium is selected from the group consisting of CIMmultus® QA-1 advanced composite column (quaternary amine), CIMmultus® DEAE-1 advanced composite column (diethylamino), CIM® QA disc (quaternary amine), CIM® DEAE, and CIM® EDA disc (ethylenediamino). In some embodiments, the monolithic anion exchange chromatography medium is a CIMmultus® QA-1 advanced composite column (quaternary amine). In some embodiments, the monolithic anion exchange chromatography medium is a CIM® QA disc (quaternary amine). In some embodiments, the anion exchange chromatography medium is CIM QA (BIA Separations, Slovenia).In some embodiments, the anion exchange chromatography medium is BIA CIM® QA-80 (column volume 80 mL). Those skilled in the art will understand that a wash buffer of suitable ionic strength can be identified so that impurities (including, but not limited to, impurities introduced by upstream purification steps) are removed while the rAAV maintains its binding to the resin.

[0179] In some embodiments, anion exchange chromatography is carried out according to the method disclosed in WO2019 / 241535 (which is incorporated herein by reference in its entirety).

[0180] In some embodiments, a method for isolating rAAV particles includes quantifying the vector genome titer, capsid titer, and / or the complete capsid:empty capsid ratio in a composition containing the isolated rAAV particles. In some embodiments, the vector genome titer is quantified by quantitative PCR (qPCR), digital PCR (dPCR), or droplet digital PCR (ddPCR). In some embodiments, the capsid titer is quantified by serotype-specific ELISA. In some embodiments, the complete capsid:empty capsid ratio is quantified by analytical ultracentrifugation (AUC) or transmission electron microscopy (TEM).

[0181] In some embodiments, the vector genome titer, capsid titer, and / or the complete capsid:empty capsid ratio are quantified by spectrophotometric measurement, for example, by measuring the absorbance of the composition at 260 nm and at 280 nm. In some embodiments, the rAAV particles are not denatured before measuring the absorbance of the composition. In some embodiments, the rAAV particles are denatured before measuring the absorbance of the composition. In some embodiments, the absorbance of the composition at 260 nm and 280 nm is quantified using a spectrophotometer. In some embodiments, the absorbance of the composition at 260 nm and 280 nm is quantified using HPLC. In some embodiments, the absorbance is peak absorbance. Several methods for measuring the absorbance of the composition at 260 nm and 280 nm are known in the art. A method for quantifying the vector genome titer and capsid titer of a composition containing isolated recombinant rAAV particles is disclosed in WO2019 / 212922 (which is incorporated herein by reference in its entirety).

[0182] In additional embodiments, the disclosure provides compositions comprising isolated rAAV particles produced according to the methods disclosed herein. In some embodiments, the compositions are pharmaceutical compositions comprising a pharmaceutically acceptable carrier.

[0183] As used herein, the term “pharmaceutically acceptable” means a bioacceptable formulation, gas, liquid, or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery, or contact. A “pharmaceutically acceptable” composition is a material that is not biologically or otherwise undesirable, for example, that can be administered to a subject without causing substantially undesirable biological effects. Such a pharmaceutical composition can therefore be used, for example, when administering rAAV isolated according to the methods of this disclosure to a subject. Such compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil in water or water in oil), suspensions, syrups, elixirs, dispersions and suspension media, coatings, isotonic and absorption enhancers or retarders, which are suitable for pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions, and suspensions may include suspending agents and thickeners. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules, and crystals. Complementary active compounds (e.g., preservatives, antimicrobial agents, antiviral agents, and antifungal agents) can also be incorporated into the composition. Pharmaceutical compositions can be formulated to be compatible with specific routes of administration or delivery, as described herein or as known to those skilled in the art. Therefore, pharmaceutical compositions may include carriers, diluents, or excipients suitable for administration via various routes.The rAAV particles, method, and suitable pharmaceutical compositions and delivery systems of the present invention are known in the art (for example, Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug Delivery Systems). (See 1980, RLJuliano, ed., Oxford, NY, pp. 253–315).

[0184] In some embodiments, the composition is a pharmaceutical unit dose. “Unit dose” means a physically separate unit suitable as a unit drug dose for the target being treated. Each unit contains a predetermined amount, optionally together with a pharmaceutical carrier (excipient, diluent, vehicle, or filler), and is calculated to produce a desired effect (e.g., prophylactic or therapeutic effect) when administered in one or more doses. Unit dosage forms may be, for example, in ampoules and vials, and these may include liquid compositions or compositions in a freeze-dried or lyophilized state, for example, by adding a sterile liquid carrier before in vivo administration or delivery. Individual unit dosage forms may be included in multi-dose kits or containers. Recombinant vectors (e.g., AAV) sequences, plasmids, vector genomes, and recombinant viral particles, as well as these pharmaceutical compositions, may be packaged in single or multiple unit dose forms to facilitate administration and ensure uniform drug dose. In some embodiments, the compositions are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, A The rAAV particles contain AAV capsid proteins from AAV capsid serotypes selected from AV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the AAV capsid serotype is AAV8. In some embodiments, the AAV capsid serotype is AAV9. [Examples]

[0185] Example 1: The AAV productivity of a process based on single-dose transient trait transfer in a 500L bioreactor was 48-60% of the productivity obtained using 50L and 200L bioreactors. The generation of adeno-associated viruses (AAVs) from cell cultures in the field of gene therapy has evolved from simple adhesive flasks to complex adhesive systems and then to suspension-based agitated tank bioreactor processes. Many AAV gene therapy generation systems exist, including transient translocation of mammalian cells (e.g., suspension-adaptive HEK293 cells). However, most of the equipment commonly used in recent suspension mammalian biotechnology processes is not specifically designed for large-scale transient generation of AAVs.

[0186] Comparing the scale and design of bioreactors expands the challenges of generating AAVs in the suspension mammalian cell translocation process. Therefore, it is necessary to compare current bioreactor systems for AAV generation. The effects of bioreactor configuration and mixing on translocation and AAV generation were compared. Bioreactors ranging from 50L to 500L were optimized for scalability and process performance. The effects of bioreactor settings and typical scale-up factors on cell growth, viability, and collection titer were compared across multiple types of bioreactors at various scales.

[0187] As substantially as described herein, AAV-A vectors (containing AAV9 capsids) were generated via transient translocation of suspension-adaptive HEK293 cells. Briefly, suspension-adaptive HEK293 cells were thawed and amplified. Approximately 1.0–1.2 × 10⁶ suspension-adaptive HEK293 cells were placed in a bioreactor. 6Cells were seeded at a density of viable cells / mL. During the 72-hour ECD (endo-collapsed culture) period, a mixture of polyethyleneimine (PEI) and three plasmids encoding adenovirus helper function, AAV-A transgene, and AAV9 Cap / Rep was transfused into the cells. Transfusion was performed using a DNA:PEI ratio of 1:1.75. Process flow diagrams for transfusion complex preparation in 20L, 200L, and 500L bioreactors are shown in Figures 1-3. DNA and PEI were mixed using an in-line mixer. The volume of the added transfusion complex was approximately 10% of the cell culture volume. The culture supernatant was collected during the 144-192 hour ECD period (e.g., 3-5 days after transfusion). The obtained AAV-A yield is shown in Figure 4. Relative titers were quantified as is customary in the art.

[0188] First-generation processes at up to 500L scale in bioreactors from multiple vendors (vendor A or B) showed that cell growth parameters were linear up to the start of generation (e.g., up to the transient translocation step) (data not shown). This indicated that P / V and Tip Speed ​​were optimal scaling parameters for cell growth. During the generation phase, cell growth and viability followed similar trends in 50L and 200L bioreactors. The collected VCD and viability of the 500L bioreactor were actually higher than those of the 50L and 200L bioreactors, indicating lower translocation efficiency. Considering that cell growth and viability were similar across multiple bioreactors tested by two different vendors, it was surprising that the productivity of the 500L bioreactor for AAV-A was only 48–60% based on the average of the past four generation runs. In contrast, the productivity of the 50L and 200L bioreactors was in line with the average. Figure 4.

[0189] Example 2: Complexation time affects AAV productivity It has been reported that the complexation time between DNA and PEI affects the size of the resulting complex, which in turn may affect the efficiency of translocation. As shown in Figure 5, longer complexation times resulted in the formation of larger DNA:PEI complexes. Complex size was measured using dynamic light scattering with known methods.

[0190] Using the experimental schematic in Figure 6, the effect of increasing complexation time on AAV-A productivity was tested. Briefly, a 42L translocation complex was prepared by mixing DNA and PEI together via an inline mixer, with PEI and a plasmid (3) encoding adenovirus helper function, AAV-A transgene, and AAV9 Cap / Rep. After mixing, the translocation complex was incubated. Then, fixed volumes of the translocation complex were taken out at predetermined time points (10, 20, 40, and 60 minutes after the start of complex incubation) and used for translocation in a bioreactor and a shaking flask.

[0191] As shown in Figure 7, AAV productivity decreased as the complex formation time increased. The results of this experiment indicate that GC titers 4 and 5 days after transfusion steadily decreased with increasing transfusion complex formation time.

[0192] Example 3: Processes based on single-dose and fractional transient translocation showed comparable AAV productivity. While not bound by any particular theory, the low productivity of AAV-A observed in Example 1 using a 500L bioreactor is thought to be due to the constraints of mixing, complexing, and adding the required volume of translocation complex (42L in the case of a 500L bioreactor) within a narrow (30-minute) operating window. As shown in Example 2, the size of the DNA:PEI complex increases over time, and using a DNA:PEI complex larger than the optimal size reduces the productivity of AAV-A. The combination of a narrow operating window of approximately 30 minutes and a large volume of translocation complex (42L) was hypothesized to lead to a low yield (50-60% of expected) due to the fact that incubation and pumping time for this large volume of translocation complex was potentially insufficient.

[0193] Processes based on fractional transient translocation were tested to determine whether the constraints on translocation complex volume and narrow operating window could be overcome by dividing the translocation process into two or more steps, such as mixing, complexing, and adding smaller volumes of translocation complex to the cell culture instead of a single large volume. The productivity of AAV-B (containing AAV8 capsid) in single-dose and fractional transient translocation-based processes was quantified using a 200 L bioreactor process. The single-dose transient translocation-based process was carried out substantially as described in Example 1. A flowchart of the translocation complex preparation process in the fractional translocation-based process is shown in Figure 8. The total volume of added translocation complex was substantially the same (approximately 16 L) in both the single-dose and fractional translocation processes. While the single-dose process involves mixing, complexing, and adding the total volume in one step, the split translocation process uses two separate mixing, complexing, and adding steps for approximately 8 L of translocation complex, with the second adding step initiated 15–30 minutes after the completion of the first adding step. Culture supernatant was collected at 168 hours of ECD, i.e., 4 days after translocation. The resulting AAV-B yield is shown in Figure 9. The split transient translocation process yielded similar titers to runs using single-dose translocation complex. This was an important finding for addressing the narrow operating window (30 minutes) in preparing and adding translocation complex volumes in large bioreactors (e.g., 200 L or more). Splitting the translocation complex into smaller doses allows for more robust complex incubation time and pump flow rate within a 30-minute operating window for each dose.

[0194] Example 4: The fractionated transdermal complex dose can be added at intervals of several hours without significantly impairing the productivity of AAV. The robustness of the fractional transient translocation process was tested using a 2L bench-scale experiment with AAV-B. The objective was to determine how long the interval between fractional translocation complex additions could be while still achieving comparable GC titers. The interval between fractional translocation complex additions was varied from 15 minutes to 6 hours. Trends in cell growth and viability were similar across all tested conditions (data not shown). Trends in glucose were also similar across conditions (data not shown). Trends in L-glutamine and NH4+ were similar across all tested conditions up to an ECD of 144 hours. Beyond this point, bioreactors with a 15-minute interval between fractional translocation complex additions consumed more L-glutamine and produced more NH4+. This difference did not negatively impact production, as these bioreactors showed comparable, or in some cases higher, GC titers than those with 4-hour and 6-hour intervals between fractional translocation complex additions. Figure 10. Although slightly larger GC titer variability was observed, segmented addition of the translocation complex, when added to the bioreactor at intervals of up to 6 hours, still yielded titers within 80–90% of the predicted range.

[0195] Example 5: The AAV-B productivity of the process based on fractional transient translocation in a 500L bioreactor is similar to that obtained using 50L and 200L bioreactors. The AAV-B productivity of a fractional transient translocation-based process in a 500L bioreactor was tested using a suspension-adaptive HEK293 clone. Figure 11 shows a flowchart of the translocation complex preparation process in the fractional translocation-based process. Two doses of approximately 21L of translocation complex were added to the culture with 15-30 minutes between additions. The culture supernatant was collected 168 hours after ECD, i.e., 4 days after translocation. Figure 12 shows the relative titer of the generated AAV-B. The productivity in the 500L bioreactor was similar to both the "historical average" and the productivity obtained using single-dose translocation processes in 200L and 50L bioreactors.

[0196] Example 6: Productivity of AAV-B process based on partial transient translocation in a 2,000L process scale-down to 200L. The AAV-B productivity of a process based on 2,000 L fractional transient translocation was tested in a 200 L bioreactor at a 200 L scale-down. An anti-aggregating agent was included in the seed train culture medium. Four doses of approximately 4 L of translocation complex were added to the culture with approximately 15 minutes between additions. The process flow diagram is shown in Figure 13. The culture supernatant was collected 168 hours after ECD, i.e., 4 days after translocation. The titer of the supernatant was approximately 6.25E+10. The solubility titer was approximately 1.15E+11.

[0197] Example 7: AAV productivity of a process based on 2,000L fractional transient translocation. In some embodiments, the process based on 2,000 L split transient translocation includes transferring 4 × approximately 42 L translocation complexes to a 2,000 L bioreactor containing approximately 1,600 L of cell culture (e.g., HEK cell culture). In some embodiments, the culture contains an anti-aggregating agent. In some embodiments, the translocation complexes are produced by mixing one or more diluted polynucleotides and at least one diluted translocation reagent using an in-line mixer, and the mixing includes transferring the one or more diluted polynucleotides and at least one diluted translocation reagent from two separate containers to a new container at a rate of approximately 8 L / min. In some embodiments, the translocation complexes are held for 10–20 minutes (e.g., 10–15 minutes) before being transferred to the bioreactor. In some embodiments, the translocation complexes are transferred to the bioreactor at a rate of approximately 5 L / min. In some embodiments, each batch of translocation complexes is transferred to the bioreactor in 30 minutes or less. In some embodiments, there may be a gap of 10–20 minutes (e.g., about 15 minutes) between the completion of transferring one batch of translocation complexes and the start of transferring the next batch.

[0198] While the methods described herein have been explained in conjunction with what is considered to be the most practical and preferred embodiments, it should be understood that the methods encompassed herein are not to be limited to the disclosed embodiments, but rather are intended to cover a variety of modifications and equivalent arrangements that fall within the spirit and scope of the appended claims.

[0199] All publications, patents, patent applications, internet sites, and accession number / database sequences (including both polynucleotide and polypeptide sequences) cited herein are incorporated herein by reference in whole for any purpose to the same extent that each individual publication, patent, patent application, internet site, or accession number / database sequence is incorporated by reference specifically and individually.

Claims

1. 1. A method for producing recombinant viral particles, comprising: a) providing between 200 liters and 20,000 liters of suspension cell culture comprising a population of cells capable of producing said recombinant viral particles; b) combining one or more polynucleotides with at least one transfection reagent to form a first mixture, incubating the mixture to form polynucleotide:transfection reagent complexes, and transferring the polynucleotide:transfection reagent complexes to the suspension cell culture for transfection into the cells; c) combining the one or more polynucleotides with the at least one transfection reagent to form a second mixture, incubating the mixture to form polynucleotide:transfection reagent complexes, and transferring the polynucleotide:transfection reagent complexes to the suspension cell culture for transfection into the cells; d) maintaining the suspension cell culture containing the transfected cells under conditions that allow the production of the recombinant viral particles; Including, (i) the one or more polynucleotides comprise genes necessary for the production of the recombinant viral particle; (ii) the combined volume of the polynucleotide:transfection reagent complexes of b) and c) transferred to the suspension cell culture is between 7% and 15% of the volume of the suspension cell culture of step a); (iii) the combining, incubating, and transferring of steps b) and c) are each completed in less than 60 minutes; (iv) the transferring of steps b) and c) is carried out over a period of not more than 6 hours; and (v) the transferring of steps b) and c) is performed simultaneously or sequentially in any order; The method.

2. 1. A method for increasing production of recombinant viral particles, comprising: a) providing between 200 liters and 20,000 liters of suspension cell culture comprising a population of cells capable of producing said recombinant viral particles; b) combining one or more polynucleotides with at least one transfection reagent to form a first mixture, incubating the mixture to form polynucleotide:transfection reagent complexes, and transferring the polynucleotide:transfection reagent complexes to the suspension cell culture for transfection into the cells; c) combining the one or more polynucleotides with the at least one transfection reagent to form a second mixture, incubating the mixture to form polynucleotide:transfection reagent complexes, and transferring the polynucleotide:transfection reagent complexes to the suspension cell culture for transfection into the cells; d) maintaining the suspension cell culture containing the transfected cells under conditions that allow the production of the recombinant viral particles; Including, (i) the one or more polynucleotides comprise genes necessary for the production of the recombinant viral particle; (ii) the combined volume of the polynucleotide:transfection reagent complexes of b) and c) transferred to the suspension cell culture is between 7% and 15% of the volume of the suspension cell culture of step a); (iii) the combining, incubating, and transferring of steps b) and c) are each completed in less than 60 minutes; (iv) the transferring of steps b) and c) is carried out over a period of not more than 6 hours; and (v) the transferring of steps b) and c) is performed simultaneously or sequentially in any order; The method. (i) step c) is repeated one more time; (ii) step c) is repeated one or more times; or (iii) step c) is repeated 1, 2, 3, 4, 5, 6, 7, or 8 times; The method according to claim 1 or 2.

4. The transferring of step c) is initiated between 5 minutes and 60 minutes after completing the transferring of step b); or The transferring of step c) is initiated within 60 minutes of completing the transferring of step b). The method according to any one of claims 1 to 3.

5. The method of any one of claims 1 to 4, wherein mixing the one or more polynucleotides with at least one transfection reagent is performed by an in-line mixer.

6. The method of any one of claims 1 to 5, wherein the combining, incubating, and transferring of steps b) and c) are each completed in less than 30 minutes or less than 35 minutes.

7. The method of any one of claims 1 to 6, wherein said incubating in steps b) and c) is for 10 to 20 minutes or 10 to 15 minutes, respectively.

8. The method of any one of claims 1 to 7, wherein said at least one transfection reagent comprises a stable cationic polymer.

9. The method of any one of claims 1 to 8, wherein said at least one transfection reagent comprises PEI.

10. The suspension cell culture is (i) having a volume between 200 liters and 5,000 liters; (ii) having a volume between 200 liters and 2,000 liters; (iii) having a volume between 200 liters and 1,000 liters; (iv) having a volume between 200 liters and 500 liters; (v) having a volume of 200 liters, 300 liters, 400 liters, 500 liters, 750 liters, 1,000 liters, 2,000 liters, 3,000 liters, or 5,000 liters; (vi) having a volume of 500 liters; (vii) has a volume of 1,000 liters; or (viii) having a volume of 2,000 liters; The method according to any one of claims 1 to 9. (i) the population of cells comprises a population of mammalian cells. (ii) the population of cells comprises a population of HEK293 cells, HEK-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, and / or PerC6 cells; (iii) the population of cells comprises a population of HEK293 cells; and / or (iv) the suspension cell culture provided in step a) contains between 2×10E+6 and 10E+7 viable cells / ml; The method according to any one of claims 1 to 10.

12. The suspension cell culture is (i) between 2 and 10 days, between 3 and 5 days, or between 5 and 14 days; (ii) 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days; (iii) for at least three days; (iv) 3 days, or (v) 4 days The method of any one of claims 1 to 11, maintained under conditions that allow the production of said recombinant viral particles.

13. The method of any one of claims 1 to 12, wherein the recombinant viral particle is a recombinant adeno-associated viral (rAAV) particle or a recombinant lentiviral particle.

14. The method of any one of claims 1 to 13, wherein the recombinant viral particle is a rAAV particle.

15. The rAAV particles are (i) comprises a capsid protein of an AAV8, AAV9, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, or AAV.hu37 serotype; and / or (ii) comprises a genome including a transgene; The method of claim 14.

16. the one or more polynucleotides a) the rAAV genome to be packaged; b) adenovirus helper functions necessary for packaging; c) AAV rep proteins sufficient for packaging, and d) AAV cap protein sufficient for packaging The method of claim 14 or 15, wherein the

17. 17. The method of claim 16, wherein the one or more polynucleotides comprise a polynucleotide encoding the rAAV genome, a polynucleotide encoding the AAV rep proteins and the AAV cap proteins, and a polynucleotide encoding the adenovirus helper functions.

18. The method of any one of claims 14 to 17, further comprising recovering the rAAV particles.

19. 19. The method of any one of claims 14 to 18, wherein the suspension cell culture produces between 5x10e+10 GC / ml and 1x10e+12 GC / ml of rAAV particles.

20. 19. The method of any one of claims 14-18, wherein the suspension cell culture produces at least twice as many rAAV particles, measured as GC / ml, as a reference method comprising a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfection reagent complex.

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